Assay plates, reader systems and methods for luminescence test measurements

ABSTRACT

Luminescence test measurements are conducted using an assay module having integrated electrodes with a reader apparatus adapted to receive assay modules, induce luminescence, preferably electrode induced luminescence, in the wells or assay regions of the assay modules and measure the induced luminescence.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/711,816, filed Dec. 12, 2019, which is a continuation of copendingU.S. patent application Ser. No. 15/587,968, filed May 5, 2017, now U.S.Pat. No. 10,539,501, which is a divisional of copending U.S. patentapplication Ser. No. 12/944,218, filed Nov. 11, 2010, now U.S. Pat. No.9,664,613, which is a divisional of copending U.S. patent applicationSer. No. 10/185,274, filed Jun. 28, 2002, now U.S. Pat. No. 7,842,246,which claims priority to U.S. Provisional Application Ser. No.60/301,932, filed Jun. 29, 2001, hereby incorporated by reference. Thisapplication is related to co-pending U.S. patent application Ser. No.10/185,363, filed Jun. 28, 2002, now U.S. Pat. No. 6,977,722, which ishereby incorporated by reference.

1. FIELD OF THE INVENTION

This application relates to plates, plate components, kits, apparatusesand methods for conducting chemical, biochemical and/or biologicalassays.

2. BACKGROUND OF THE INVENTION 2.1 Chemical, Biochemical and BiologicalAssays

Numerous methods and systems have been developed for conductingchemical, biochemical and/or biological assays. These methods andsystems are essential in a variety of applications including medicaldiagnostics, food and beverage testing, environmental monitoring,manufacturing quality control, drug discovery and basic scientificresearch.

Depending on the application, it is desirable that assay methods andsystems have one or more of the following characteristics: i) highthroughput, ii) high sensitivity; iii) large dynamic range, iv) highprecision and/or accuracy, v) low cost, vi) low consumption of reagents,vii) compatibility with existing instrumentation for sample handling andprocessing, viii) short time to result, ix) insensitivity tointerferents and complex sample matrices and x) uncomplicated format.There is substantial value to new assay methods and systems thatincorporate improvements in these characteristics or in otherperformance parameters.

At this time, there are a number of commercially available instrumentsthat utilize electrochemiluminescence (ECL) for analytical measurements.Species that can be induced to emit ECL (ECL-active species) have beenused as ECL labels. Examples of ECL labels include: i) organometalliccompounds where the metal is from, for example, the noble metals ofgroup VIII, including Ru-containing and Os-containing organometalliccompounds such as the tris-bipyridyl-ruthenium (RuBpy)moiety and ii)luminol and related compounds. Species that participate with the ECLlabel in the ECL process are referred to herein as ECL coreactants.Commonly used coreactants include tertiary amines (e.g., see U.S. Pat.No. 5,846,485, herein incorporated by reference), oxalate, andpersulfate for ECL from RuBpy and hydrogen peroxide for ECL from luminol(see, e.g., U.S. Pat. No. 5,240,863, herein incorporated by reference).The light generated by ECL labels can be used as a reporter signal indiagnostic procedures (Bard et al., U.S. Pat. No. 5,238,808, hereinincorporated by reference). For instance, an ECL label can be covalentlycoupled to a binding agent such as an antibody or nucleic acid probe;the participation of the binding reagent in a binding interaction can bemonitored by measuring ECL emitted from the ECL label. Alternatively,the ECL signal from an ECL-active compound may be indicative of thechemical environment (see, e.g., U.S. Pat. No. 5,641,623 which describesECL assays that monitor the formation or destruction of ECL coreactants,herein incorporated by reference). For more background on ECL, ECLlabels, ECL assays and instrumentation for conducting ECL assays seeU.S. Pat. Nos. 5,093,268; 5,147,806; 5,324,457; 5,591,581; 5,597,910;5,641,623; 5,643,713; 5,679,519; 5,705,402; 5,846,485; 5,866,434;5,786,141; 5,731,147; 6,066,448; 6,136,268; 5,776,672; 5,308,754;5,240,863; 6,207,369; and 5,589,136 and Published PCT Nos. WO99/63347;WO00/03233; WO99/58962; WO99/32662; WO99/14599; WO98/12539; WO97/36931and WO98/57154, each of which are herein incorporated by reference.

Commercially available ECL instruments have demonstrated exceptionalperformance. They have become widely used for reasons including theirexcellent sensitivity, dynamic range, precision, and tolerance ofcomplex sample matrices. The commercially available instrumentation usesflow cell-based designs with permanent reusable flow cells. The use of apermanent flow cell provides many advantages but also some limitations,for example, in assay throughput. In some applications, for example, thescreening of chemical libraries for potential therapeutic drugs, assayinstrumentation should perform large numbers of analyses at very highspeeds on small quantities of samples. A variety of techniques have beendeveloped for increasing assay throughput. The use of multi-well assayplates allows for the parallel processing and analysis of multiplesamples distributed in multiple wells of a plate. Typically, samples andreagents are stored, processed and/or analyzed in multi-well assayplates (also known as microplates or microtiter plates). Multi-wellassay plates can take a variety of forms, sizes and shapes. Forconvenience, some standards have appeared for some instrumentation usedto process samples for high throughput assays. Multi-well assay platestypically are made in standard sizes and shapes and having standardarrangements of wells. Some well established arrangements of wellsinclude those found on 96-well plates (12×8 array of wells), 384-wellplates (24×16 array of wells) and 1536-well plate (48×32 array of well).The Society for Biomolecular Screening has published recommendedmicroplate specifications for a variety of plate formats (see,http://www.sbsonline.org), the recommended specifications herebyincorporated by reference.

Assays carried out in standardized plate formats can take advantage ofreadily available equipment for storing and moving these plates as wellas readily available equipment for rapidly dispensing liquids in and outof the plates. A variety of instrumentation is commercially availablefor rapidly measuring radioactivity, fluorescence, chemiluminescence,and optical absorbance in or from the wells of a plate, however, thereis no commercial instrument for measuring ECL emitted from the wells ofa multi-well assay plate.

2.2 Assay Plates

FIG. 1 depicts a standard 96-well assay plate 100. Assay plate 100comprises a skirt 112, a periphery wall 114, a upper surface 116 and an8×12 array of wells 118 separated by spacers 120 and empty base regions128. Skirt 112 surrounds the base of plate 100 and typically has a widthof 3.365 inches and a length of 5.030 inches. To facilitate orientation,skirt 112 and periphery wall 114 include a recess 130. Upper surface 116extends around plate 100 from periphery wall 114 to respective midlinesof the outermost wells of wells 118. Each of wells 118 comprises a cellwall 122 having an inner surface 124 and a cell floor 126, togetherdefining a cylindrical region. Skirt 112, periphery wall 114, uppersurface 116, wells 118, spacers 120, cell floors 126 and base regions128 are integrally molded features of plate 100. Alternatively, plate100 may omit cell floors 126.

A standard 96-well assay plate is not particularly suited forelectrochemiluminescence test measurements. The small size of the wellsin such a plate, approximately 0.053 square inches each, presents aconsiderable obstacle for the introduction of electrodes and/or theefficient collection of light emitted from the surface of suchelectrodes. The dimensional problems grow even more difficult whenplates having even higher well concentrations are considered, e.g.384-well plates and 1536-well plates.

3. SUMMARY OF THE INVENTION

The invention relates to assay modules (preferably assay plates, morepreferably multi-well assay plates), methods and apparatuses forconducting assay measurements. Assay modules of the invention mayinclude one or more, preferably a plurality, of wells, chambers and/orassay regions for conducting one or more assay measurements. Preferably,these wells, chambers and/or assay regions comprise one or moreelectrodes for inducing luminescence from materials in the wells,chambers and/or assay regions. The assay modules may further compriseassay reagents (in liquid or dry form), preferably in the wells,chambers or assay regions of the assay module. Such assay reagents maybe immobilized on electrodes of the module or confined on electrodes ofthe module (e.g., through the use of appropriately designed dielectricsurfaces surrounding the electrode surfaces). Preferably, the module isconfigured to allow for the measurement of luminescence in portions ofthe assay module (preferably, more than one assay region, well orchamber at a time, but less than all). One aspect of the inventionrelates to novel configurations and materials for electrodes andelectrical contacts in assay modules. The invention also relates toapparatuses, methods, systems and kits for conducting measurements usingassay modules. The invention further relates to methods of manufacturingthe assay modules and plates of the invention.

The multi-well assay plates may include several elements, for example, aplate top, a plate bottom, wells, working electrodes, counterelectrodes, reference electrodes, dielectric materials, electricalconnections, and assay reagents. The wells of the plates may be definedby holes/openings in the plate top. The plate bottom can be affixed tothe plate top (either directly or in combination with other components)and can serve as the bottom of the well. Alternatively, the wells of theplates may be defined as indentations or dimples on a surface of aplate. The multi-well assay plates may have any number of wells of anysize or shape, arranged in any pattern or configuration, and can becomposed of a variety of different materials. Preferred embodiments ofthe invention use industry standard formats for the number, size, shapeand configuration of the plate and wells. Examples of standard formatsinclude 96-, 384-, 1536-, and 9600-well plates, with the wellsconfigured in two-dimensional arrays. Other formats may include singlewell plates (preferably having a plurality of assay domains), 2 wellplates, 6 well plates, 24 well plates, and 6144 well plates.

According to the invention, working, counter and, optionally, referenceelectrodes can be incorporated into the wells. The present inventiondescribes several novel configurations and materials for electrodes inmulti-well assay plates. Multi-well assay plates of the presentinvention may be used once or may be used multiple times and are wellsuited to applications where the plates are disposable. Furthermore, theassay reagents, preferably dried reagents and/or wet reagents, may beincorporated into the assay plate, preferably into one or more wells orassay domains. In some embodiments, a well of a multi-well plate mayinclude a plurality of assay domains.

The invention relates to processes that involve the use of an electrodeand the generation of light, including methods, apparatuses and assaymodules adapted for such processes. The invention further relates to themeasurement of light from such processes, for example, in the conduct ofassays. Examples of such processes include electrochemiluminescence(also referred to as electrogenerated chemiluminescence),electroluminescence, and chemiluminescence triggered by anelectrochemically generated species. For the purposes of the applicationand for convenience, these three processes will be referred to as“electrode induced luminescence”. Electrochemiluminescence involveselectrogenerated species and the emission of light. For example,electrochemiluminescence may involve luminescence generated by a processin which one or more reactants are generated electrochemically andundergo one or more chemical reactions to produce species that emitslight, preferably repeatedly. The invention also relates to processesthat do not require the use of an electrode, for example,chemiluminescence, fluorescence, bioluminescence, phosphorescence,optical density and processes that involve the emission of light from ascintillant. The invention also relates to processes that do not involveluminescence, for example, electrochemical processes (e.g., involvingthe measurement or generation of current or voltage) or electricalprocesses (e.g., involving the measurement of resistance or impedance).

The invention further relates to an apparatus that can be used to induceand measure luminescence, preferably electrode induced luminescence,more preferably electrochemiluminescence, in assays conducted in or onassay modules, preferably multi-well assay plates. The invention furtherrelates to an apparatus that can be used to conduct assays by certainoptically based assay methodologies that do not use electrode inducedluminescence such as fluorescence assays, chemiluminescence assays,bioluminescence assays and phosphorescence assays. The invention alsorelates to an apparatus that can be used to induce and/or measurecurrent and/or voltage, for example, at an electrode. The measurement ofcurrent and/or voltage may occur independently of or concurrently withillumination and/or with the measurement of luminescence (e.g., as inspectroelectrochemical measurements or photoelectrochemicalmeasurements).

The apparatus may incorporate, for example, one or more photodetectors;a light tight enclosure; mechanisms to transport the assay plates intoand out of the apparatus (and in particular, into and out of a lighttight enclosure); mechanisms to align and orient the assay plates withthe photodetector(s) and/or with electrical contacts; mechanisms totrack and identify plates (e.g. bar code readers); mechanisms to makeelectrical connections to plates, one or more sources of electricalenergy for inducing luminescence, and appropriate devices, electronicsand/or software. The apparatus may also include mechanisms to store,stack, move and/or distribute one or more multi-well assay plates (e.g.plate stackers and/or plate conveyors). The apparatus may be configuredto measure light from multi-well assay plates by measuring lightsequentially from a plurality of sectors of the plate and/or from theentire plate substantially simultaneously or simultaneously. Theapparatus may also incorporate microprocessors and computers to controlcertain functions within the instrument and to aid in the storage,analysis and presentation of data.

Another aspect of the invention relates to methods for performing assayscomprising measuring luminescence from an assay plate. According to thepresent invention, luminescence is advantageously measured from theassay plate in sectors. Another embodiment relates to methods forperforming electrode induced luminescence (preferablyelectrochemiluminescence) assays in a multi-well plate having aplurality of wells.

Yet another aspect of the invention relates to assay plates and platecomponents (e.g., plate bottoms, plate tops and multi-well plates) foruse in a variety of assays. Thus, one embodiment relates to platebottoms (e.g., without the plate top) which can be joined with a platetop to form a multi-well plate suitable for assays. For example, a platebottom having a plurality of patterned electrodes which may be on a topsurface, the electrodes arranged in such a manner so that when thebottom is joined with a multi-well plate top, each well has one or more,preferably two or more, conductive electrode surfaces.

Another embodiment relates to an improved plate top having one or moreopenings configured so that when affixed or placed onto a plate bottomforms one or more assay wells. Preferably, the plate top forms wellshaving well surfaces with properties and characteristics (e.g., lightreflection, surface tension, etc.) for improved assays. For example,plate tops designed to form well surfaces which provide for improvedluminescence collection efficiencies.

A still further aspect of the invention relates to systems comprisingthe apparatus of the present invention combined with the multi-wellplate of the invention. Preferably, the system contains all thecomponents necessary for performing assays such as high-throughputassays including a light detector, a source of electrical energy and aplate support with a multi-well plate placed thereon.

A still further aspect of the invention relates to kits for use in theassay plates, apparatuses and methods of the invention. Preferably, thekits include, in one or more containers, a multi-well plate and one ormore assay reagents.

4. DESCRIPTION OF THE FIGURES

FIG. 1 illustrates an industry standard multi-well assay plate having 96wells.

FIG. 2K illustrates a top view of a multi-well assay plate according toone embodiment of the invention.

FIG. 2A illustrates a top view of a well 200 of a multi-well assay plateaccording to another embodiment of the invention. Well 200 has a wall162, having an inner surface 164; a counter electrode 166; and a workingelectrode 168 which forms the bottom of well 200.

FIG. 2B illustrates a top view of a well 220 of a multi-well assay plateaccording to the invention. Well 220 has a wall 222, counter electrodes226A and 226B and a working electrode 230.

FIG. 2C illustrates a top view of a well 240 of a multi-well assay plateaccording to the invention. Well 240 has wall 242, counter electrodes246A and 246B, and working electrode 250.

FIG. 2D illustrates a top view of a well 260 of a multi-well assay plateaccording to the invention. Well 260 has wall 262, counter electrode266, and working electrode 270.

FIG. 2E illustrates a plate top 280 according to the present invention.Plate top 280 comprises a plate top body 281, a top surface 282, wall285, and inner surface 286. Plate top 280 has holes 284 that may be usedin part to form walls for wells in multi-well assay plates of theinvention.

FIG. 2F illustrates a plate top 290 according to the present inventionwherein plate top 290 has 384 square holes 291.

FIG. 2G illustrates a plate top 295 according to the invention whereinplate top 295 has 1536 holes 297.

FIG. 2H illustrates a cross sectional view from the side of a multi-wellassay plate 2000 according to the present invention. Plate 2000 has asupport/working electrode 2001, a plurality of wells 2002, dielectriclayer 2004, counter electrode 2006, lip 2008 and plate top 2009. Plate2000 may also incorporate other features described elsewhere formulti-well assay plates such as assay reagents, electrical connections,supporting materials, etc.

FIG. 2I illustrates cross-sectional view from the side of a multi-wellassay plate 2010 of the invention. Multi-well assay plate 2010 has asupport 2011, one or more wells 2012, working electrode 2013, dielectriclayer 2014, counter electrode 2016, lip 2018 and boundary 2019. Plate2010 may also incorporate other features described elsewhere formulti-well assay plates such as assay reagents, electrical connections,supporting materials, etc.

FIG. 2J illustrates a cross sectional view from the side of a multi-wellassay plate 2020 of the invention. Plate 2020 has a support 2021, one ormore wells 2022, one or more working electrodes 2023, one or morecounter electrodes 2026, lip 2028 and one or more boundaries 2029. Plate2020 may also incorporate other features described above for multi-wellassay plates such as assay reagents, electrical connections, supportingmaterials, etc.

FIG. 3A illustrates a well 300 according to another embodiment of thepresent invention. Well 300 has a wall 302 having an interior surface304, counter electrodes 306A and 306B, working electrode 310 and assaydomains 312.

FIG. 3B illustrates a well 330 according to the present inventionwherein well 330 has a plurality of assay domains 336.

FIG. 3C illustrates a well 360 according to the present inventionwherein well 360 has a plurality of assay domains 366.

FIG. 4A illustrates a well 400 according to yet another embodiment ofthe present invention. Well 400 has a wall 402 having an interiorsurface 404, counter electrodes 406A and 406B, working electrode 410,and boundaries 416 that define domains 418 of working electrode 410.

FIG. 4B illustrates a well 430 according to the invention. Boundary 440separates counter electrodes 434A and 434B from working electrode 444.

FIG. 4C illustrates a well 460 according to the invention whereinboundary 470 separate counter electrodes 464A and 464B from workingelectrode 474. Working electrode 474 has a plurality of assay domains476.

FIG. 4D illustrates a well 480 according to the invention with a wall482, counter electrodes 488A and 488B, boundary 492, working electrode494, boundaries 498A and 498B and assay domains 499A and 499B.

FIG. 4E illustrates a well 4900 according to the present invention. Well4900 has wall 4902 with interior surface 4903, counter electrodes 4904Aand 4904B, gaps 4906A and 4906B exposing a support, barrier 4908 with aplurality of holes 4912 that expose working electrode 4910.

FIG. 5 illustrates a multi-well assay plate 500 according to anotherembodiment of the invention.

FIGS. 6A-6C illustrate examples of sectioned conductive layers inmulti-well assay plate of the invention. FIG. 6A shows a conductivelayer 600 sectioned into six sections 602A, 602B, 602C, 602D, 602E, and602F. FIG. 6B shows conductive layer 620 sectioned into 12 sections622A-L. FIG. 6C shows conductive layer 640 shows sectioned into 96sections 644.

FIGS. 7A-7D illustrate examples of sectioned electrodes in multi-wellassay plate of the invention. FIG. 7A shows electrode 700 sectioned intosix sections. FIG. 7B shows electrode 720 sectioned into 12 sections.FIG. 7C shows electrode 740 sectioned into 8 sections. FIG. 7D showselectrode 760 sectioned into 96 sections.

FIG. 8A illustrates a multi-well assay plate 800 of the invention.

FIG. 8B illustrates a multi-well assay plate 830 of the invention.

FIG. 8C illustrates a stylized cross sectional view of two wells 842Aand 842B from the multi-well assay plate 830 shown in FIG. 8B.

FIG. 9A shows the components of a multi-well plate 930 according to theinvention.

FIG. 9B shows a stylized cross sectional view of three wells from themulti-well assay plate 930 shown in FIG. 9A.

FIG. 10A illustrates a multi-well assay plate 1000 of the invention.

FIG. 10B shows a stylized cross sectional view of three wells from themulti-well assay plate 1000 shown in FIG. 10A.

FIG. 11A illustrates a 96-well assay plate 1100 of the invention.

FIG. 12A illustrates a 384-well assay plate 1200 of the invention.

FIG. 13A illustrates a multi-well assay plate 1300 of the invention thathas multiple fluid containment regions in each well.

FIG. 13B illustrates a multi-well assay plate 1350 of the invention thathas multiple fluid containment regions in each well.

FIG. 14A illustrates a multi-well assay plate 1400 of the invention thathas multiple fluid containment regions in each well.

FIG. 14B shows a stylized cross sectional view of three wells from the96-well assay plate 1400 shown in FIG. 14A.

FIG. 15 illustrates a multi-well assay plate 1500 of the invention thathas multiple fluid containment regions in each well.

FIG. 16A illustrates a multi-well assay plate 1600 of the inventionhaving a single patterned conductive layer on a substrate. FIG. 16Bshows a conductive layer 1640 having a working electrode section 1642and counter electrode section 1644. FIG. 16C shows plate 1660 anddemonstrates alternative schemes for sectioning electrodes in multi-wellassay plates.

FIG. 17 illustrates an apparatus according to one embodiment of thepresent invention. Reader 1700 comprises a cover 1702, a light tightenclosure 1704 with one or more doors or apertures 1714, a photodetector1706, optics 1708, multi-well assay plate 1710, plate aligner 1712,plate transporter 1716, bar code reader 1718, electronics 1720,current/voltage source 1722, plate electrical connector 1724, computer1726, power supply 1728, data and network connections 1730, indicators1732, reagent handler 1734, one or more plate stackers 1736, robotics1738, and plate carrier 1740.

FIG. 18 illustrates an apparatus according to the present invention.Reader 1800, which shows selected elements, illustrates a light tightenclosure 1804, photodetector 1806, optics 1808, plate transporter 1816,plate electronics 1820, input plate stacker 1836A, output plate stacker1836B, input plate stack 1837A, output plate stack 1837B, and outputdoor or aperture 1814B.

FIG. 19 illustrates selected components of an apparatus according to thepresent invention wherein the illustration highlights the alignment ofoptics 1908, photodetector 1907, plate sector 1910A, and plateelectrical connector 1924 having contacts 1925. Light tight enclosure1904, door or aperture 1914, plate 1910, plate carrier 1940 and platetransporter 1916 are also present.

FIG. 20 illustrates selected components of an apparatus according to thepresent invention wherein the illustration highlights the imaging of asector 2042A of a multi-well assay plate 2042 of the invention.Photodetector 2057, optics 2058, filter 2059, plate carrier 2040 andplate transporter 2047 are also indicated.

FIG. 21 illustrates selected components of an apparatus of the inventionwherein the illustration highlights the relative positions of platesector 2110A, plate electrical connector 2124 with contacts 2125, andphotodiode array 2107 of photodetector 2106. Plate 2110, photodetectorcircuit board 2105, plate transporter 2116, and plate carrier 2140 arealso shown.

FIG. 22 illustrates selected components of an apparatus of the inventionwherein the illustration highlights photodiode array 2207 where therelative positions of photodiodes 2207A-H with wells 2210A-Hrespectively of multi-well assay plate 2210. Plate electrical connector2224, electronics 2220, electrical contacts 2205, shield 2208, lighttight enclosure 2204 and plate carrier 2240 are also shown.

FIG. 23 illustrates an apparatus according to the present invention.Reader 2300, which shows selected elements, illustrates a chassis 2301,photodetector 2306, multi-well assay plate 2310, plate transporter 2316,plate electrical connector 2324 and a plurality of contacts 2325.

FIG. 24 shows the ECL signal emitted from wells of several embodimentsof the multi-well assay plates of the invention as a function of theconcentration of ruthenium-tris-bipyridine in the wells. The ECL signalwas measured by imaging using a cooled CCD camera.

FIG. 25 shows the ECL signal emitted from wells of two embodiments ofthe multi-well assay plates of the invention as a function of theconcentration of ruthenium-tris-bipyridine in the wells. The ECL signalwas measured with an array of eight photodiodes.

FIG. 26 demonstrates the use of two embodiments of multi-well assayplates of the invention for carrying out sandwich immunoassays forprostate specific antigen (PSA). The plot shows the ECL signal as afunction of the concentration of PSA. The ECL signal was measured byimaging with a cooled CCD camera.

FIG. 27 demonstrates the use of two embodiments of multi-well assayplates of the invention for carrying out sandwich immunoassays for PSA.The plot shows the ECL signal as a function of the concentration of PSA.The ECL signal was measured with an array of eight photodiodes.

FIG. 28 demonstrates the use of three embodiments of multi-well assayplates of the invention for carrying out sandwich immunoassays for AFP.The plot shows the ECL signal as a function of the concentration of AFP.The ECL signal was measured by imaging with a cooled CCD camera.

FIG. 29 demonstrates the independent measurement by ECL sandwichimmunoassay of four analytes (IL-1β, IL-6, TNF-α and IFN-γ) in wells ofa multi-well assay plate. The working electrode in each well ispatterned with four assay domains, each assay domain comprising acapture antibody specific for one of the analytes. The plots show theECL signal emitted from each assay domain as a function of theconcentration of each analyte.

FIG. 30 demonstrates the independent measurement by ECL sandwichimmunoassay of four analytes (IL-1β, IL-6, TNF-α and IFN-γ) in wells ofa multi-well assay plate. The working electrode in each well ispatterned with four assay domains, each assay domain comprising acapture antibody specific for one of the analytes. The figure shows animage of the ECL emitted from a sector of wells used to assay samplescontaining varying mixtures of the four analytes. The highlighted wellis annotated to show the arrangement of the four assay domains. Thatspecific well was used to assay a sample having 250 pg/mL each of IL-1βand TNF-α and 8 pg/mL each of IL-6 and IFN-γ.

FIG. 31 demonstrates the use of multi-well assay plates of the inventionfor carrying out a nucleic acid hybridization assay. The plot shows theECL signal as a function of the concentration of aruthenium-tris-bipyridine labeled DNA target. The ECL signal wasmeasured by imaging with a cooled CCD camera.

FIG. 32 demonstrates the use of a multi-well assay plate of theinvention in a chemiluminescence-based assay.

FIG. 33 shows the integrated electrochemiluntinescence intensity emittedfrom a 1536-well plate of the invention as function of the concentrationof ruthenium(II)-tris-bipyridine dichloride in the wells.

FIG. 34A shows preferred contact locations on assay plate having a 2×3array of six square sectors.

FIG. 34B shows preferred contact locations on assay plate having anarray of 12 columnar sectors.

FIG. 35a-f is a representative block diagram of an automated diagnosticdevice utilizing a CCD camera.

FIG. 36a-f is a representative block diagram of an automated diagnosticdevice utilizing a photodiode array.

FIGS. 37A-37C is a diagram illustrating placement of bar codeinformation along the edges of a microtiter plate.

FIG. 38 illustrates a top view of a four spot well configured to bemeasured by a single light detector according to one embodiment of theinvention.

5. DETAILED DESCRIPTION OF THE INVENTION

The invention includes instrumentation and methods for conducting avariety of different types of measurements. The word “measurement” andverb forms of “to measure” as used herein include both quantitative andqualitative determinations.

The invention includes assay modules (e.g., plates, dipsticks,measurement cells, cassettes, cartridges, elements or devices), plate ormodule components, apparatuses and methods for performingluminescence-based assays. The present invention describes several novelconfigurations and/or materials for electrodes in assay modules,particularly multi-well assay plates. One embodiment relates to an assaymodule having a plurality of assay domains or assay regions and,preferably, one or more wells or chambers. The assay modules of thepresent invention may be used once or may be used multiple times; inpreferred embodiments, the modules (e.g., plates) are disposable.

In this specification, inventive concepts may be disclosed in thecontext of assay plates (e.g., preferred electrode configurations,electrode materials, laminar structures, means for making electricalcontacts to an electrode from the bottom of a plate, apparatuses andmethods for measuring electrode induced luminescence (preferablyelectrochemiluminescence)), however, the concepts are also applicable toembodiments relating to other types of assay modules. The preferredembodiments of the invention relate to assay modules, preferably assayplates, having a plurality of assay wells (e.g., “multi-well plates”).Preferred apparatus of the invention are designed to operate with themulti-well assay modules and generally incorporates features forinducing and measuring electrode induced luminescence. The multi-wellassay modules and apparatus of the present invention greatly improveamong other things the speed, efficiency, quality, ease and cost ofluminescence, particularly electrode induced luminescence, moreparticularly electrochemiluminescence, measurements.

The multi-well assay modules (e.g., plates) of the invention enable theperformance of electrode induced luminescence-based assays inside one ormore wells or chambers of a multi-well assay module (e.g., the wells ofa multi-well assay plate). Multi-well assay plates may include severalelements including, for example, a plate top, a plate bottom, wells,working electrodes, counter electrodes, reference electrodes, dielectricmaterials, contact surfaces for electrical connections, conductivethrough-holes electrically connecting the electrodes and contactsurfaces, adhesives, assay reagents, and identifying markings or labels.The wells of the plates may be defined by holes in the plate top; theinner walls of the holes in the plate top may define the walls of thewell. The plate bottom can be affixed to the plate top (either directlyor in combination with other components) and can serve as the bottom ofthe well.

The multi-well assay modules (e.g., plates) may have any number of wellsand/or chambers of any size or shape, arranged in any pattern orconfiguration, and be composed of a variety of different materials.Preferred embodiments of the invention are multi-well assay plates thatuse industry standard multi-well plate formats for the number, size,shape and configuration of the plate and wells. Examples of standardformats include 96-, 384-, 1536- and 9600-well plates, with the wellsconfigured in two-dimensional arrays. Other formats include single well,two well, six well and twenty-four well and 6144 well plates.Preferably, the wells and/or chambers have at least one first electrodeincorporated therein, and more preferably also include at least onesecond electrode. According to preferred embodiments, the wells and/orchambers have at least one working electrode incorporated therein, andmore preferably also include at least one counter electrode. Accordingto a particularly preferred embodiment, working, counter and,optionally, reference electrodes are incorporated into the wells and/orchambers. The assay plates are preferably flat, but may also be curved(not flat).

Moreover, one or more assay reagents may be included in wells, chambersand/or assay domains of an assay module (e.g., in the wells of amulti-well assay plate). These assay reagents may be immobilized orplaced on one or more of the surfaces of a well and/or chamber(preferably on the surface of an electrode, most preferably a workingelectrode) and may be immobilized or placed in one or more distinctassay domains (e.g. in patterned arrays of reagents immobilized on oneor more surfaces of a well and/or chamber, preferably on workingelectrodes and/or counter electrodes, most preferably on workingelectrodes). The assay reagents may also be contained or localized byfeatures within the well and/or chamber. For example, patterneddielectric materials may confine or localize fluids.

The preferred apparatus of the invention can be used to induce andmeasure luminescence in assays conducted in assay modules, preferably inmulti-well assay plates. It may incorporate, for example, one or morephotodetectors; a light tight enclosure; electrical connectors forcontacting the assay modules; mechanisms to transport multi-well assaymodules into and out of the apparatus (and in particular, into and outof light tight enclosures); mechanisms to align and orient multi-wellassay modules with the photodetector(s) and with electrical contacts;mechanisms to track and identify modules (e.g. one or more bar codereaders (e.g., one bar code reader for reading one side of a plate ormodule and another for reading another side of the plate or module);orientation sensor(s); mechanisms to make electrical connections tomodules, one or more sources of electrical energy for inducingluminescence in the modules; and appropriate electronics and software.

The apparatus may also include mechanisms to store, stack, move and/ordistribute one or more assay modules (e.g. multi-well plate stackers).The apparatus may advantageously use arrays of photodetectors (e.g.arrays of photodiodes) or imaging photodetectors (e.g. CCD cameras) tomeasure light. These detectors allow the apparatus to measure the lightfrom multiple wells (and/or chambers) simultaneously and/or to image theintensity and spatial distribution of light emitted from an individualwell (and/or chamber).

The apparatus can preferably measure light from one or more sectors ofan assay module, preferably a multi-well assay plate. In someembodiments, a sector comprises a group of wells (and/or chambers)numbering between one and a number fewer than the total number of wells(and/or chambers) in the assay module (e.g. a row, column, ortwo-dimensional sub-array of wells in a multi-well plate). In preferredembodiments, a sector comprises between 4 percent and 50 percent of thewells of a multi-well plate. In especially preferred embodiments,multi-well assay plates are divided into columnar sectors (each sectorhaving one row or column of wells) or square sectors (e.g., a standardsized multi-well plate can be divided into six square sectors of equalsize). In some embodiments, a sector may comprise one or more wells withmore than one fluid containment region within the wells. The apparatus,preferably, is adapted to sequentially induce ECL in and/or sequentiallymeasure ECL from the sectors in a given module, preferably plate.

The apparatus may also incorporate microprocessors and computers tocontrol certain functions within the instrument and to aid in thestorage, analysis and presentation of data. These microprocessors andcomputers may reside in the apparatus, or may reside in remote locationsthat interact with the apparatus (e.g. through network connections).

In a general description of a preferred measurement operation, samples,reactants, and reagents for electrode induced luminescence (preferablyelectrochemiluminescence) assays are introduced into assay modules(preferably, into one or more wells of multi-well assay plates). Themodules (e.g., the plates and the contents of their wells) areintroduced into the measurement apparatus, either one at a time, or inmultiples (e.g., by using a plate stacker). A module is, preferably,transported into an enclosed region of the apparatus and, in particular,into a light-tight enclosure. The apparatus positions the module so thatone or more (preferably, one) sectors are in alignment with thephotodetector(s) and/or with electrical connector mechanisms. Aftermaking electrical contact to a sector, the apparatus applies a voltageand/or current waveform and induces luminescence from labels within thatsector. The apparatus measures the emitted light with photodetector(s)and stores the results. The apparatus may then sequentially repeat themeasurements on other sectors (preferably, one sector at a time). Thesequential measurement of sectors may involve making electrical contactto a plurality of sectors and then sequentially applying electricalenergy to the appropriate sectors and/or it may involve moving themodule, photodetector(s) and/or electrical contacts with respect to eachother so as to align the photodetectors and/or electrical contacts withthe appropriate sector before firing. In an alternate embodiment, theapparatus may be adapted to measure the entire module at once. After allmeasurements are complete, the module is then, preferably, transportedout of the light-tight enclosure.

In particularly preferred embodiments, the assay modules (in particular,the multi-well assay plates) and apparatus according to the presentinvention can greatly improve the speed and efficiency with whichluminescence measurements may be conducted. By incorporating the abilityto induce electrode induced luminescence directly in a well of amulti-well assay plate, the invention overcomes an important limitationof the prior art, namely, the need to transfer the contents of a well ina standard multi-well plate (which lacks the features necessary forelectrode induced luminescence tests) into a separate instrument thatcan conduct electrode induced luminescence-based measurements. Inpreferred examples of the present invention, multiple electrode inducedluminescence (preferably electrochemiluminescence) test measurements maybe conducted in different wells of the same plate simultaneously. Suchsimultaneous operation dramatically increases the rate at which samplesmay be processed, eliminates cross-contamination of samples,significantly improves overall testing efficiency and enables themeasurement of multiple analytes simultaneously. Because the preferredembodiments of the present invention incorporate electrodes into eachwell of the multi-well assay plates, it eliminates the need for apermanent, reusable measurement cell in the apparatus, whichsignificantly reduces the cost and complexity of the apparatus. Bymeasuring luminescence from sectors in a multi-well assay plate, theapparatus balances the desirable characteristics of rapid measurementtimes and high optical collection efficiencies.

An important advantage of the multi-well assay plates according to thepresent invention is the ability to make them compatible with otherapparatus already adapted to handle industry-standard multi-well plates.Compatibility with existing plate handling equipment facilitates rapid,efficient and economic loading, processing, storage and disposal ofassay plates. Standard plate handling equipment may be used to transportassay plates from one apparatus to another or to and from storage.Existing fluid transfer equipment, such as automatic pipettingequipment, plate washers and mixing stations may be used to transfersamples, reactants, solutions and other reagents to and from theindividual wells of a multi-well assay plate. Advantageously, the shapeand size of the assay plates is compatible with standard apparatuses forthe conduct of pre-processing reactions, shaking or mixing operations orstorage. Compatibility with existing equipment and sample handlingprocesses allow for ready integration of the multi-well assay plates andapparatus of the present invention with existing laboratory equipmentfor handling and processing plates (such equipment may be incorporated,in whole or in part, into the apparatus and/or functionally linked oradjoined to the apparatus). This compatibility may be particularlyadvantageous in high throughput screening operations.

5.1 Multi-Well Assay Plates

One aspect of the invention relates to improved assay modules (e.g.,plates) adapted for use in assays, preferably luminescence assays, morepreferably electrode induced luminescence assays, even more preferablyelectrochemiluminescence assays. The assay modules of the invention arepreferably suitable not only for ECL assays, but also suitable forfluorescence assays, chemiluminescence assays, bioluminescence assays,phosphorescence assays, optical transmittance assays (e.g., measurementsof optical density or light scattering) and electrochemical assays(e.g., wherein the measurement involves measuring current or voltage).

According to one preferred embodiment of the invention, an assay moduleor plate comprises one or more (preferably two or more, 6 or more, 24 ormore, 96 or more, 384 or more, 1536 or more or 9600 or more) assaywells, assay chambers and/or assay domains (e.g., discrete locations ona module surface where an assay reaction occurs and/or where an assaysignal is emitted; typically an electrode surface, preferably a workingelectrode surface). According to a particularly preferred embodiment,the assay plate is a multi-well assay plate having a standard wellconfiguration (e.g., 6 well, 24 well, 96 well, 384 well, 1536 well, 6144well or 9600 well).

An electrode induced luminescence well (preferablyelectrochemiluminescence well (i.e., a well adapted forelectrochemiluminescence)) or electrode induced luminescence domain(preferably electrochemiluminescence assay domain (i.e., an assay domainadapted for electrochemiluminescence assays)) may include a firstelectrode surface (such as a working electrode surface) and, preferablyalso includes a second electrode surface (such as a counter electrodesurface).

The invention also relates to a multi-well module, preferably an assayplate, for conducting one or more assays, the module having a pluralityof wells (and/or chambers), wherein two or more of the plurality ofwells (and/or chambers) comprise at least one first electrode surfaceand, preferably at least one counter electrode surface. According to apreferred embodiment, two or more of the plurality of wells (and/orchambers) comprise a working electrode surface and, preferably a counterelectrode surface, adapted to induce luminescence in the wells. Theinvention also relates to a multi-well module, preferably a plate, forconducting one or more assays, the module having a plurality of wells,wherein one or more of the plurality of wells comprise a workingelectrode surface and a counter electrode surface adapted to induceluminescence in the wells. Preferably, all or substantially all of thewells comprise an electrode surface.

Another embodiment relates to a multi-well assay module, preferably anassay plate, for conducting electrode induced luminescence (preferablyelectrochemiluminescence) assays, the module, preferably plate, having aplurality of wells, wherein each of the plurality of wells comprises atleast one first electrode surface (e.g., a working electrode) and,preferably, at least one second electrode surface (e.g., a counterelectrode).

Another embodiment relates to an assay plate for conducting one or moreelectrode induced luminescence (preferably electrochemiluminescence)assays, the plate having a plurality of wells or assay regionscomprising electrode surfaces, wherein the electrode surfaces consistessentially of at least one working electrode surface and at least onecounter electrode surface.

Preferably, the assay regions or assay wells are free of referenceelectrodes allowing for a greater density of assay domains andsimplified instrumentation for inducing and measuring luminescence.

Preferably, the working electrode is adjacent, but not physicallycontacting the counter electrode. Preferably, the working electrodesurface and counter electrode surface are at substantially the sameheight or at the same height within the well.

According to another embodiment, the spacing between the workingelectrode and counter electrode is preferably small, more preferablyless than 0.5 inch, even more preferably less than 0.2 inch, even morepreferably less than 0.1 inch, even more preferably less then 0.05, evenmore preferably less than 0.01 inch and most preferred less than 0.005inch. Preferably, the electrodes are integrated into the assay module,preferably assay plate, allowing luminescence, preferably electrodeinduced luminescence, more preferably electrochemiluminescence, to beinduced without the use of an external electrode probe. Preferably, anassay reagent is immobilized on the working electrode (discussed furtherbelow). In another preferred embodiment no assay reagent is immobilizedon the working electrode (discussed further below). In yet anotherpreferred embodiment, one or more assay reagents are immobilized on theworking electrode (discussed further below). In yet another preferredembodiment, two or more assay reagents are immobilized on the workingelectrode (discussed further below).

In order to enhance luminescence collection efficiency and/or reduce thesize of the imaging surface and/or number of light detectors, the moduleis preferably electrically addressable in sectors. That is, rather thanmeasuring light from a single well, chamber, or assay domain at a time(which is time inefficient) or measuring light from the entire module(which reduces light collection efficiencies, requires multiple lightdetectors or requires the use of larger light detectors), the module andapparatus are configured to allow for the measurement of luminescence inportions of the assay module (preferably, more than one assay domain,well or chamber at a time, but less than all). Preferably, the portionsof the assay module are in sectors, where the terms “sector” or“sectors” when used in the context of a plate or module is used hereinto refer to independently addressable groups of one or more (preferablytwo or more) jointly addressable assay wells, assay chambers or assaydomains. Preferably, the sectors comprise one or more electrodes, morepreferably two or more jointly addressable (e.g., electricallyconnected) working electrodes.

One embodiment relates to an assay module (preferably, an assay plate,more preferably a multi-well plate) for conducting luminescence assays(preferably electrode induced luminescence assays, more preferablyelectrochemiluminescence assays) comprising a substrate surface having aplurality of electrodes patterned thereon, wherein the plurality ofelectrodes are patterned so as to form independently addressable sectorscomprising jointly addressable electrodes.

According to another embodiment, the assay module (preferably amulti-well plate) has a plurality of wells, each well comprising a firstelectrode surface (preferably suitable for use as a working electrode inan electrode induced luminescence assay) and, preferably, a secondelectrode surface (preferably suitable for use as a counter electrode inthe electrode induced luminescence assay). Referring to FIG. 2K, eachwell 158 of multi-well assay plate 150 according to a particularlypreferred embodiment of the invention comprises a working electrode 168and a counter electrode 166.

The working electrode surface area may be smaller, the same or largerthan the counter electrode surface area. In some embodiments, theworking electrode surface is preferably much larger than the counterelectrode surface. See FIGS. 2A, 2B and 2D, for example. Thisconfiguration allows for a greater working electrode surface on which toimmobilize assay reagents. Preferably, the surface ratio of the workingelectrode surface to the counter electrode surface is at least 2 to 1,more preferably at least 5 to 1, even more preferably at least 10 to 1,still more preferred at least 50 to 1, even more preferably at least 100to 1 and most preferred at least 500 to 1. Surprisingly, the assaymodules of the invention provide for the performance ofelectrochemiluminescence assays with very little counter electrodesurface. Preferably, the working electrode is substantially centeredwithin the well so as to maximize the percentage of ECL emitted from theelectrode that can be captured by a light detector placed above thewell.

According to another embodiment, the first electrode surface (e.g.,working electrode surface) is centered at the bottom of each well andthe second electrode surface (e.g., counter electrode surface) isadjacent the periphery of the bottom of each well. In some embodiments,the working electrode surface is centered at the bottom of each well andis completely surrounded by the counter electrode surface. Referring toFIG. 2D, working electrode 270 is completely surrounded by counterelectrode 266. Preferably, the counter electrode surface is adjacent,but not in contact, with the working electrode (being separated by gapand/or insulating material 268).

Another embodiment of the invention relates to a multi-well assay modulehaving a plurality of wells, each well having a well bottom comprising afirst electrode surface, a second electrode surface and a dielectricsurface (preferably the dielectric surface is the surface of the bottomof the well between the first electrode surface and the second electrodesurface), wherein the ratio of the first electrode surface and thedielectric surface is at least 5 to 1, preferably 10 to 1, morepreferably 30 to 1.

According to another embodiment the well bottom comprises 30 to 99.1%working electrode surface, 0.1 to 50% counter electrode surface and 0.01to 70% dielectric surface. Preferably, the well bottom comprises 30 to99.1% working electrode surface, 0.1 to 30% counter electrode surfaceand 0.01 to 70% dielectric surface, more preferably the well bottomcomprises 50 to 99.1% working electrode surface, 0.1 to 20% counterelectrode surface and 0.01 to 70% dielectric surface, even morepreferably 75 to 99.1% working electrode surface, 0.1 to 10% counterelectrode surface and 0.01 to 70% dielectric surface, even morepreferably 80 to 99.1% working electrode surface, 0.1 to 5% counterelectrode surface and 0.01 to 70% dielectric surface and most preferably85 to 99.1% working electrode surface, 0.1 to 1% counter electrodesurface and 0.01 to 70% dielectric surface.

Alternatively, for some applications it is desirable that workingelectrode surfaces be small, e.g., relative to the surface area of awell or well bottom. In some applications, this configuration may reducenon-specific signals. According to one embodiment of the invention, themulti-well assay module has a plurality of wells, each well having awell bottom comprising a first electrode surface, a second electrodesurface and a dielectric surface (preferably the dielectric surface isthe surface of the bottom of the well between the first electrodesurface and the second electrode surface), wherein the ratio of thefirst electrode surface and the dielectric surface (or alternatively thesurface of the well bottom) is less than 1 to 5, preferably 1 to 10,more preferably 1 to 30.

According to one preferred embodiment of the invention, the assay modulecomprises a first electrode surface (preferably a working electrodesurface) that is bounded by a dielectric surface, the dielectric surfacebeing raised or lowered (preferably, raised) and/or of differenthydrophobicity (preferably, more hydrophobic) than the electrodesurface. Preferably, the dielectric boundary is higher, relative to theelectrode surface, by 0.5-100 micrometers, or more preferably by 2-30micrometers, or most preferably by 8-12 micrometers. Even morepreferably, the dielectric boundary has a sharply defined edge (i.e.,providing a steep boundary wall and/or a sharp angle at the interfacebetween the electrode and the dielectric boundary). Preferably, thefirst electrode surface has a contact angle for water 10 degrees lessthan the dielectric surface, preferably 15 degrees less, more preferably20 degrees less, more preferably degrees less, even more preferably 40degrees less, and most preferred 50 degrees less. One advantage ofhaving a dielectric surface that is raised and/or more hydrophobic thanthe electrode surface is in the reagent deposition process where thedielectric boundary may be used to confine a reagent within the boundaryof the electrode surface. In particular, having a sharply defined edgewith a steep boundary wall and/or a sharp angle at the interface betweenthe electrode and dielectric boundary is especially useful for “pinning”drops of solution and confining them to the electrode surface.

According to another embodiment, an assay module comprises one or more(preferably two or more) wells, the wells having one or more firstelectrode surfaces (preferably one or more working electrode surfaces)and a plurality of assay domains immobilized therein. Preferably, atleast two of the plurality of the assay domains comprises differentbinding reagents. Preferably, each well comprises at least four, morepreferably at least seven, even more preferably at least ten assaydomains and most preferred at least 15 assay domains. One preferredembodiment is a 24 well plate wherein each well comprises at least 16,preferably at least 25, more preferably at least 64, even morepreferably at least 100 assay domains per well and most preferably atleast 250 assay domains per well.

Another embodiment of the invention relates to a multi-well module(preferably a multi-well plate) having a plurality of wells, wherein thewells comprise a plurality of working electrode surfaces having assaydomains immobilized thereon. Preferably, the assay domains areindependently addressable. For example, a well may comprise a pluralityof assay domains, wherein each assay domain comprises an electrode whichis independently addressable from the other assay domains within thewell. In another example, a group of wells may each comprise a pluralityof assay domains, wherein each assay domain comprises an electrode whichis independently addressable from the other assay domains within thewell, but which is jointly addressable with an assay domain in each ofthe other wells.

As discussed above and described in more detail below, one aspect of theinvention may involve detecting emitted luminescence using an imagingsystem. According to a preferred embodiment, the apparatus may employ acamera, which images the assay module (e.g., a multi-well plate). Sincethe distance between the camera or imaging surface and the source ofluminescence (e.g., working electrode surface) can impact the quality ofthe image, controlling such distances is preferred. For example, if theworking electrode surfaces (e.g., the surfaces at which luminescence maybe induced or generated) are formed on well bottoms and two or morewells are imaged simultaneously, the height of the working electrodesurface (and corresponding distance to the camera) is preferablysubstantially the same. Preferably, the variation is less than 0.01inches, more preferably less than 0.005 inches and most preferably lessthan 0.001 inches. Thus, the parameters, which may cause such variation,are preferably controlled (e.g., electrode thickness and height, flexingor warping of the assay module, etc.).

Thus, the plate bottom of an assay plate is preferably flat. Forexample, when a multi-well assay plate is placed on a flat surface, thevariation in height measured from the flat surface to the electrodesurfaces in each of the plurality of wells is preferably less than 0.01inches, more preferably less than 0.005 inches and most preferably lessthan 0.001 inches. That is, referring to the cross-sectional view inFIGS. 2H, 2I and 2J and FIGS. 8C, 9B, 10B and 14B, the vertical heightof each working electrode surface in each of the wells is preferablysubstantially the same (i.e., the same vertical height throughout thewell or assay region). Preferably, the vertical height within at leastthe wells within each sector is the same (i.e., the same vertical heightthroughout the sector). Even more preferably, the vertical height withineach sector of a plate is substantially the same (i.e., the samevertical height throughout the plate). Otherwise, the light detector orimaging system may need to be re-focused for each sector to optimize themeasurement (discussed further below in Section 5.8).

Accordingly, another embodiment relates to a multi-well platecomprising:

-   -   (a) a plurality of wells, the wells having well bottoms; and    -   (b) a plate substrate;

wherein when the multi-well plate is placed on a flat surface, the wellbottom is elevated from the flat surface 0.050 to 0.150 inches,preferably, 0.103 to 0.107 inches, more preferably 0.104-0.106, and mostpreferred about 0.105.

Providing a more uniform and consistent well bottom elevation enablescontrol of the electrode surface height variation, even for differentplate formats. Preferably, the plate comprises greater than 100 wells orless than 90 wells. Thus, the height may be maintained whether the plateis a 96 well plate, a 6 well plate, a 384 well plate or otherwise. Thisallows for the use of different plate configurations without distortingthe image or without having to refocus the imaging system. That is, onemay use a variety of different plate formats without re-focusing theimaging system if the distance between the camera and working electrodesurface is maintained from plate to plate. This is particularlyadvantageous, for example, if a plate stack including a number of plateshaving different plate formats is being used.

Preferably, the plate bottom has a thickness less than 10 cm, preferablyless than 5 cm, even more preferably less than 1 cm, even morepreferably less than 5 mm, even more preferably less than 1 mm, evenmore preferably 0.1 mm, even more preferably 0.01 mm, and most preferred0.001 mm.

According to one embodiment, the plate bottom elevation is providedusing “legs” or a skirt to elevate the plate off any surface on which itrests. FIG. 1 illustrates skirt 112 and FIG. 8C illustrates skirt 836,both of which are embodiments of “skirts” according to the inventionthat may be configured to elevate the plate. Preferably, the plate iselevated to maintain the distance between the working electrode surfaceand the imaging surface or camera. Thus, although the well depth of the384 well plate may be different than that of a 96 well plate, the legson the 96 well plate would be configured to adjust its working electrodesurface to be comparable to that of the 384 well plate. Advantageously,the skirt and/or the elevation of the plate bottom are also configuredso as to prevent contact between top edge or lip of the well of oneplate in a stack with the bottom surface of the next higher plate in thestack. Preventing such contact prevents the plates from stickingtogether and reduces condensation from occurring on the bottom ofplates. Alternatively, the plates may be adapted to form a seal whenstacked (e.g., to reduce or prevent contamination and/or the evaporationof the well contents).

According to one embodiment, an adhesive layer 944 may be employed toboth attach a plate top to a plate bottom and also provide sealingbetween the wells. (See also, for example, adhesive layer 806 of FIG.8A; adhesive layer 844 of FIG. 8B; adhesive layer 1030 of FIG. 10A;adhesive layer 1530 of FIG. 15; and adhesive layer 1604 of FIG. 16A).Preferably, the thickness of the adhesive layer is 0.0002-0.01 inches,more preferably 0.0005-0.008 inches, even more preferably 0.002-0.006inches and most preferably approximately 0.005 inches. Preferably, insuch embodiments, the well walls are at least 0.03 inches, or morepreferably, at least 0.05 inches thick to allow for reliable andleak-free sealing. According to one preferred embodiment, the adhesivelayer is a double coated film preferably comprising at least a 0.5 mil(0.02 mm) carrier film (e.g., polyester) coated on both sides with anadhesive (preferably an acrylic based adhesive) of at least 2 mils (0.08mm). The carrier provides dimensional stability and the 2 mil adhesivecoat prevents leaks. Preferably, the adhesive layer is Keystone TapesW-546, 3M 4768 or a combination thereof, more preferably 3M 4768. Othersuitable adhesives or adhesive layers may include Ideal (887), 3M (444,442, 415), Morgan IB-2100, Nashua 943, Permacel P-941, Tesa 4972, AveryDennison adhesives (e.g., UVA tape) and Adhesives Research adhesives.

Preferably, the wells are separated from each adjacent well by between0.03 and 0.3 inches, preferably (for 96 well plates) between about 0.09and 0.11 inches, most preferred about 0.104 inches. Optimizing the wellwall thickness and well separation advantageously reduces and preferablyprevents sample leakage from one well into another well. This may be aproblem, for example, if the electrical contacts of the apparatus pushup onto the well bottoms causing flexing.

Another way to mitigate the problem of well leakage involves improvingthe sealing between the wells. Referring to FIG. 9B, working electrodesurface 958 and dielectric layer 950 preferably extend beyond well 942.Thus, one embodiment of the invention relates to a multi-well platecomprising a dielectric surface and a working electrode layer, whereinthe dielectric surface is comprised of a dielectric layer formed on aportion of the working electrode layer wherein the working electrodelayer and the dielectric layer extend beyond the well walls. Accordingto another embodiment, the working electrode layer and the dielectriclayer are deposited onto a plate bottom or substrate and extend beyondthe well helping to seal the wells. Preferably, at least a portion ofthe working electrode surface, the counter electrode surface, and/or thedielectric layer extend beyond the well wall.

According to another aspect of the invention, one or more of theelectrodes are integrated into a plate bottom or assay module substrate.In one embodiment of the invention, an assay module is formed bycombining such a plate bottom or assay module substrate with a suitableassay module top. The top may comprise holes, wells, channels, tubes,compartments, etc. that define wells, chambers, channels, tubes and/ormicrofluidics within the assay module. Thus, the invention also relatesto plate bottoms or assay module substrates having a variety ofelectrodes, electrical contacts and conductive through-holecombinations. Also included within the scope of the invention aremulti-well plates, formed by attaching a suitable plate top to the platebottom and apparatuses and methods adapted to perform assays using suchplates.

Thus, another aspect of the invention relates to assay modulesubstrates, preferably multi-well plate bottoms (e.g., having no platetop). For example, such plate bottoms can be affixed with the plate topthus forming a multi-well plate for use in conducting assays.

FIG. 2K illustrates a multi-well assay plate 150 according to apreferred embodiment of the present invention. A 96-well assay plate 150comprises an outer lip 152, an inner lip 154, a top surface 156, and 96individual wells 158 separated by spacers 160. Defined between wells 158and spacers 160 are inter-well regions 170.

Preferably, the majority of plate 150 (e.g., all but the bottom surfacesof wells 158) is a unitary molded structure made from rigidthermoplastic material such as polystyrene, polyethylene orpolypropylene (alternatively, the entire plate, including the bottomsurface of wells 158, may be a unitary structure). According to onepreferred embodiment, the material comprises polystyrene blended withHigh Impact Polystyrene (HIPS) to reduce the brittleness of thematerial. Preferably, between 4 and 16 wt % HIPS is blended with thepolystyrene, more preferably between about 8 and 12 wt %. Optimally, theunitary structure of plate 150 is formed of inexpensive material that isgenerally impervious to reagents typically encountered in ECLmeasurements, resistant to the adsorption of biomolecules, and canwithstand modest levels of heat and light. Advantageously, the platematerials (including any adhesives used to seal the wells) areimpervious to organic solvents typically used to dissolve chemicallibraries for high throughput screening (preferably the plate isunaffected by 10% aqueous solutions of DMSO or methanol, more preferablyby 20% aqueous solutions of DMSO or methanol, or most preferably by 100%DMSO or methanol). Preferably the use of silicone-containing materialsis avoided in the components used to make up a plate since silicones cancontaminate surfaces of the plate and affect wetting, adsorptive and/orelectrode properties of surfaces (preferably, the plate or a givencomponent of the plate contains less than 1 wt % silicone, morepreferably less than 0.1 wt % silicone or, most preferably, less than0.01% silicone).

Different colored material for plate 150 may be used to improve theresults of certain ECL measurement processes. It is preferable to use amaterial that does not transmit light so as to prevent cross-talkbetween wells. A highly reflective metallic coating or constituentmaterial may provide an especially reflective interior surface for eachof wells 158 to increase the efficiency with which light can betransmitted to photodetectors. An opaque white plastic material such asa plastic filled with light scattering particles (e.g., lead oxide,alumina, silica or, preferably, titanium dioxide particles) may providean interior surface for each of wells 158 that is highly lightscattering thereby improving light gathering efficiency. Alternatively,an opaque black material for plate 150 may advantageously prevent thereflection or scattering of ECL-generated light from different locationswithin a well 158 so as to prevent reflective interference during ECLtest measurements. In general, when imaging light emitted from a well(e.g., when using a camera to produce an image of light emitted from thewell) it is advantageous that the interior surface of wells 158 comprisean absorptive (e.g., black) non-scattering material since the detectionof scattered light will reduce the fidelity of the image. In general,when detecting light in a non-imaging mode (e.g., when a single lightdetector is used to detect all the light emitted from a well) it isadvantageous that the interior surface of wells 158 comprise areflective or highly scattering material so as to maximize thecollection of light at the detector.

Plate 150 may be composed of several parts joined together. In manyembodiments plate 150 and elements outer lip 152, inner lip 154, topsurface 156, spacers 160, inter-well region 170, corner recesses orchamfers 172, and wall 162 (having interior surface 164) comprise aplate top. The plate top of plate 150 may have holes, the sides of whichare defined by interior surface 164 of wall 162. The plate top can thenbe combined with a plate bottom that defines, together with the platetop, wells 158. The plate bottom advantageously comprises a workingelectrode 168 and may further comprise a counter electrode 166. Theplate bottom may, optionally, comprise one or more independent referenceelectrodes (not shown). Preferably, reference electrodes are notincluded. The plate bottom may be a continuous element or may becomposed of many elements, either coupled together or completelydistinct. Working electrode(s) 168 may comprise the predominantstructure for the plate bottom or, alternatively, may be supported onanother element that provides appropriate structural properties. Theplate bottom may be affixed to the plate top by a variety of means, forexample, by using adhesives or other bonding agents, conducting ordielectric films, by bonding, fusing or welding the constituent parts,by mechanical fasteners such as clamps, screws, tabs and slots, or byother structures or means known in the art.

Alternatively, plate 150 may be formed from any material that can beformed into an appropriate shape. Materials such as plastics,elastomers, ceramics, composites, glasses, metals, carbon materials orthe like can be used. While it is preferred that the majority of plate150 be a single unitary structure, it is within the scope of the presentinvention to provide plate 150 with removable or otherwise contiguablecomponents, particularly wells 158. Plate 150 can be conductive ornon-conductive. In applications in which plate 150 is conductive, plate150 may be grounded or itself function as a counter electrode or aworking electrode.

Outer lip 152 extends downwardly and inwardly to provide a rigid lipextending around the entire periphery of plate 150. Outer lip 152 mayfunction to aid in the alignment and orientation of plate 150 and mayfunction to allow robotic systems to handle the plate. As shown, outerlip 152 preferably includes two recessed corner recesses 172 thatprovide identifying physical indicia for plate 150. In particular,corner recesses 172 facilitate the alignment and handling of plate 150and assist in distinguishing plate 150 from other plates havingdifferent configurations of recessed areas along their respectiveperipheries. Advantageously, the dimensions and structure of outer lip152 are preferably in accordance with, or at least compatible with,industry standards for the footprints of similar types of assay plate.

Inner lip 154 extends upwardly from the top surface of outer lip 152 toa height slightly above top surface 156. Top surface 156 is thusrecessed within inner lip 154. The otherwise rectangular shape of innerlip 154 is interrupted at two corners by corner cutouts shaped to definepart of corner recesses 172.

Top surface 156 extends around the periphery of plate 150 within theconfines of inner lip 154. Preferably, top surface 156 extends inward tothe mid point of each of the outer most of wells 158. Alternatively, topsurface 156 is a continuous surface extending throughout the areasdefined between wells 158. As preferred, spacers 160 structurallyconnect wells 158 to each other and, in conjunction with the outsidesurfaces of wells 158, define inter-well regions 170.

Each of wells 158, preferably, comprise a wall 162, an interior surface164, a counter electrode 166, and a working electrode 168. As shown,wall 162 may define a cylindrical volume extending above top surface 156and downwardly to at least counter electrode 166 or working electrode168. Alternatively, wall 162 may not extend to electrodes 166 or 168 andmay be flush with top surface 156. In another embodiment, not shown,wall 162 is rectilinear with a quadrilateral cross-sectional, preferablysquare or rectangular.

Wall 162 has an interior surface 164 that is preferably cylindrical inshape and defines a volume of well 158. Inner surface 164 preferablyextends the depth of well 158. At bottom, or at a position near thebottom of well 158, counter electrode 166 and/or working electrode 168comprise a bottom surface of well 158. Such bottom surface is preferablynot integral to plate 150 or well 158 in that it is formed of differentmaterials. Counter electrode 166 and working electrode 168 may becoplanar or at different depths within well 158. Preferably, interiorsurface 164, counter electrode 166 and working electrode 168 togetherform a container suitable for holding liquids as well as solids, gelsand similar states of matter.

Inter-well regions 170 may be open passages extending through plate 150or, preferably, include base structure integral to plate 150. Such basestructure may bear indicia identifying wells 158 individually. Inaddition to corner recesses 172, plate 150 may bear other identifyingindicia. For example, plate 150 may include a bar code identificationstripe pattern on top surface 156, on the exterior of inner lip 154, onthe exterior surface of outer lip 152, on the underside of plate 150, orelsewhere on plate 150.

In alternate preferred embodiments, plate 150 may be configured as amulti-well assay plate having any number of wells. For example, 1-well,6-well, 24-well, 96-well, 384-well, 1536-well, 6144-well and 9600-wellplates may be constructed in accordance with the present invention asdescribed herein. Multi-well assay plates of the invention may have anumber of wells ranging from 1 to 2, 2 to 6, 6 to 24, 24 to 96, 96 to384, 384 to 1536, 1536 to 9600, 6144 to 100,000, or greater than100,000. Preferred embodiments have wells that range in volume from nLto 100 nL, 100 nL to 1 uL, from 1 uL to 100 uL, from 100 uL to 1 mL andfrom 500 uL to 10 mL. The wells 158 of plate 150 may be configured inmany different shapes and sizes, such as wells with rectangular crosssections, very shallow wells or depressions (dimples) or the like, toaccommodate particular reaction criteria or existing equipment andimplement integrated ECL electrode technology according to the presentinvention.

Assay reagents (e.g., binding reagents, coreactants, ECL labels) may beimmobilized on the bottom surface of the well 158. These reagents may becovalently or non-covalently immobilized on the bottom surface.Advantageously, reagents are immobilized on the working electrode 168.In preferred embodiments, assay reagents are immobilized in assaydomains on working electrode 168. These assay domains may be distinct orcontiguous. In some embodiments, multiple distinct assay domainscontaining assay reagents are present on the working electrode 168.

According to one embodiment, the plate further comprises a cover or lidor plate seal (“cover”) adapted to cover the wells and thereby reduce orprevent evaporation and/or prevent contamination. The cover may be, forexample, a hard plastic cover or an adhesive flexible tape. The covermay be disposable and/or reusable.

According to one embodiment, the cover is opaque to protect lightsensitive components within the plate. In this embodiment, the cover isremoved prior to measurement of the luminescence. According to anotherembodiment, the cover is transparent, preferably transparent enough toallow luminescence to be measured through it. Preferably, at least thebottom surface of the cover is treated (e.g., with a hydrophilic orhydrophobic coating) to prevent detrimental clouding of the lid.According to one embodiment, the bottom surface is hydrophobic to reducecondensation and thereby reduce clouding. According to an alternativeembodiment, the bottom surface is hydrophilic to promote uniform wettingand thereby also reduce clouding.

5.1.1 Embodiments of Wells in a Multi-Well Assay Plate

FIGS. 2A-D, 3A-C, and 4A-E provide views of a number of alternativeconfigurations for wells 158. These figures show wells having firstelectrodes (preferably working electrodes), second electrodes(preferably counter electrodes), and in some cases, boundaries. Thefigures show the exposed surfaces of the components. In some embodimentsof the invention some of these components may have additional surfacesburied under other components. Preferably, the first and secondelectrodes are not in electrical contact (e.g., there is a least a smallgap or some interposing material between the electrodes in the verticaland/or horizontal dimensions). In the figures, a line shown dividingworking and counter electrode surfaces may represent such a gap orinterposing material. Preferably, the working electrode is placed at orsubstantially at the center of the well bottom, so as to minimizeshadowing by the well walls of luminescence generated at the workingelectrode; preferably, the counter electrode is placed at orsubstantially at the edges of the wells. FIG. 2A illustrates well 200comprising wall 162, having an inner surface 164; a counter electrode166; and a working electrode 168. Well 200 may, optionally, comprise areference electrode (not shown). As shown, interior surface 164 of wall162 defines a cylindrical volume. At or near the bottom of suchcylindrical volume, counter electrode 166 extends in a ring-shape areabetween interior surface 164 and circular working electrode 168. Wall162 is preferably comprised of materials described previously for plates150. It may also, however, be comprised of other materials and/or havecoatings on its surface 164. Counter electrode 166 may be coplanar withthe surface of working electrode 168 or it may be at a different depth.It may also be a material or coating affixed to inner surface 164. It ispreferred that counter electrode 166 itself defines a cylindrical volumeabove working electrode 168. Preferably, counter electrode 166 has aninner radius which is at least 20% of the radius of inner surface 164,more preferably is at least 50% of the radius of inner surface 164 andmost preferably is at least 80% of the radius of inner surface 164.Preferably, counter electrode 166 is not in direct electrical contactwith working electrode 168 and a gap or insulating layer (not shown) isinterposed between electrodes 166 and 168.

FIG. 2B illustrates well 220, another embodiment of well 158. Well 220comprises wall 222 having an interior surface 224; counter electrode226A and 226B; and working electrode 230. Preferably, counter electrodes226A and 226B are symmetrical electrode areas abutting opposite sides ofinterior surface 224. Preferably, counter electrodes 226A and 226B areelectrically isolated from working electrode 230 by a gap or insulatinglayer interposed between the electrodes. Preferably, each of counterelectrodes 226A and 226B is less than 40%/of the cross-sectional areadefined by inner surface 224, more preferably is less than 20% of sucharea, and most preferably is less than 10% of such area. Well 220 may,optionally, comprise a reference electrode (not shown).

FIG. 2C illustrates well 240, another embodiment of well 158. Well 240comprises wall 242 having an interior surface 244, counter electrodes246A and 246B, and working electrode 250. Preferable counter electrodes246A and 246B abut opposite sides of interior surface 244. Preferably,counter electrodes 246A and 246B are electrically isolated from workingelectrode 250 by a gap (or insulating layer) 248A and 248B interposedbetween the electrodes. Preferably, each of counter electrodes 246A and246B is less than 40% of the cross-sectional area defined by innersurface 244, more preferably is less than 20% of such area, and mostpreferably is less than 10% of such area. Well 240 may, optionally,comprise a reference electrode (not shown).

FIG. 2D illustrates well 260, another embodiment of well 158. Well 260comprises wall 262 having an interior surface 264, counter electrode266, and working electrode 270. Preferable counter electrode 266 abutsinterior surface 264. Preferably, counter electrode 266 is electricallyisolated from working electrode 270 by a gap (or insulating layer) 268interposed between the electrodes. Preferably, counter electrode 266 isless than 40% of the cross-sectional area defined by inner surface 264,more preferably is less than 20% of such area, even more preferably lessthan 10% of such area, even more preferably less than 5% of such areaand most preferably is less than 1% of such area. Well 260 may,optionally, comprise a reference electrode (not shown).

FIG. 3A illustrates well 300, another embodiment of well 158. Well 300comprises wall 302 having an interior surface 304, counter electrodes306A and 306B and working electrode 310. Well 300 may, optionally,comprise a reference electrode (not shown). Preferably counterelectrodes 306A and 306B abut interior surface 304. Counter electrodes306A and 306B are preferably electrically isolated from workingelectrode 310 by a gap (or insulating layer) 308A and 308B interposedbetween the electrodes. Working electrode 310 may be, but is notnecessarily, in contact with interior surface 304. Working electrode 310has one or more assay domains 312. Assay domains 312 may contain assayreagents. Preferably assay domains 312 comprise assay binding reagents(so as to form binding domains), reaction substrates (e.g., substratesof enzymatic activities) or calibration reagents. Assay domains 312 maycomprise assay reagents in dry, liquid, gel or solid form. The reagentsmay be immobilized on working electrode 310. Assay domains 312 maycomprise binding reagents for one or more analytes in a sample, and eachassay domain may contain the same or different assay reagents. Assaydomains 312 may be formed by depositing reagents (e.g., by a variety ofmethods understood for depositing reagents) on specified locations onthe surface of working electrode 310 or may be incorporated into workingelectrode 310 (e.g., as reagents entrained in the material that composesworking electrode 310). In another embodiment, assay domains 310 aredefined as regions of working electrode 310 with different physical,chemical or compositional properties relative to each other and/or toother regions of the surface of working electrode 310. For example,assay domains may represent especially hydrophilic or hydrophobicregions, regions of high or low surface area, depressions orprotrusions, regions surrounded by physical barriers and/or regions ofhigh or low conductivity. They may also comprise regions with one ormore materials (e.g., a gel) deposited on the surface of the electrode.FIGS. 3B and 3C illustrate wells 330 and 360 respectively, which showadditional embodiments. Well 330 has assay domains 336 which arearranged in a different pattern than assay domains 312. Well 360 hasassay domains 366 which illustrate different shapes for assay domains366. It will be appreciated that the shape, number, pattern ofdistribution and properties of assay domains as described herein canhave many variations, all of which are encompassed by the presentinvention. Preferably, each well comprises one or more, preferably atleast two domains, more preferably at least four, even more preferablyat least seven, even more preferably at least 15 and most preferably atleast 20 assay domains. Other preferred embodiments include plateswherein each well comprises at least 50, more preferably at least 75,even more preferably at least 100 assay domains per well.

FIGS. 4A through 4E show embodiments of well 158 that illustrate the useof boundaries to define one or more distinct exposed regions and/orassay domains on a working electrode. While each figure shows specificnumbers, shapes and arrangements of the exposed regions or assaydomains, it is understood that the invention encompasses wells varyingin these parameters. FIG. 4A illustrates well 400, another embodiment ofwell 158, and shows the use of boundaries to form distinct regions on anelectrode (in particular, assay domains as described above). Well 400comprises wall 402 having an interior surface 404, counter electrodes406A and 406B and working electrode 410. Well 400 may, optionally,comprise a reference electrode (not shown). Preferably counterelectrodes 406A and 406B abut interior surface 404. Counter electrodes406A and 406B are electrically isolated from working electrode 410 by agap (or insulating layer) 408A and 408B interposed between theelectrodes. Working electrode 410 may be, but is not necessarily, incontact with interior surface 404. Working electrode 410 has a pluralityof regions 420, each having an inner region 418 defined by boundaries416 (in an alternate embodiment well 400 has only one region 420).Boundary 416 may be comprised of a material deposited on workingelectrode 410 or may be comprised of the same material as workingelectrode 410. Boundary 416 may be a region in which material has beenremoved from working electrode 410. Boundary 416 may also compriseregions of working electrode 410 with different physical, chemical orcompositional properties. For example, boundary 416 may, relative tointerior regions 416, be hydrophilic or hydrophobic, have high or lowsurface area, have a different height and/or have a high or lowconductivity. Preferably, boundary 416 is composed of non-conducting ordielectric materials deposited on the surface of working electrode 410.Boundary 416 may be coplanar with working electrode 410 and may extendout from or into the surface of working electrode 410. Inner regions 418may comprise assay domains as described above. In preferred embodiments,boundary 416 confines materials (e.g. liquids, assay reagents, and thelike) on working electrode 410. Boundaries 416 may be used to aid in ordirect the deposition of materials to regions 420, for example, bypreventing spreading of liquids deposited in inner regions 418 tosurrounding regions of working electrode 410 or to counter electrodes406A and 406B (e.g., so as to allow the controlled immobilization ofreagents onto defined assay domains on working electrode 410). In oneembodiment, the meniscus of fluids or other materials confined withinboundary 416 may act as a lens. Boundary 416 may also serve as anindicia during measurements using the apparatus of the presentinvention, e.g., to allow the location or identification of an assaydomain. FIG. 4B illustrates well 430, another embodiment of well 158.Well 430 comprises wall 431 having an interior surface 432, counterelectrodes 434A and 434B and working electrode 444. Well 430 may,optionally, comprise a reference electrode (not shown). The exposedregion of working electrode 444 is defined by boundary 440 having aninner perimeter and an outer perimeter (alternatively, a plurality ofholes in boundary 440 may define a plurality of exposed regions ofworking electrode 444). Boundary 440 may abut counter electrodes 434Aand 434B and may abut interior surface 432. Alternatively, boundary 440may extend below counter electrodes 434A and 434B or, alternatively, atleast partially above counter electrodes 434A and 434B. In someembodiments, boundary 440 extends beneath counter electrodes 434A and434B and electrically isolates them from working electrode 444. Boundary440 may be comprised of a material deposited on working electrode 444and may or may not be comprised on the same material as workingelectrode 444. Boundary 440 may be a region in which material has beenremoved from working electrode 444. Boundary 440 may also comprise aregion of working electrode 444 with different physical, chemical orcompositional properties. Preferably, boundary 440 is composed of adielectric material deposited on the surface of working electrode 444.Boundary 440 may be coplanar with working electrode 444 and may extendout from or into the surface of working electrode 444. Working electrode444 may have assay domains as described above. In preferred embodiments,boundary 440 confines materials (e.g. liquids, assay reagents, and thelike) on working electrode 444 (e.g., so as to allow the controlledimmobilization of reagents onto working electrode 444). Boundary 440 maybe used to aid in or direct the deposition of materials to workingelectrode 444, for example, by preventing spreading of liquids tosurrounding regions of working electrode 444 or to counter electrodes434A and 434B. Boundary 440 may also serve as indicia duringmeasurements using the apparatus of the present invention. In apreferred embodiment: working electrode 444 is a conducting material,either self supporting or supported on another material; boundary 440 isa non-conducting material deposited on working electrode 444 that coversworking electrode 444 except in regions defined by the inner perimeterof boundary 440; counter electrodes 434A and 434B are deposited onboundary 440 and are electrically isolated from working electrode 444 byboundary 440; and wall 431 with interior surface 432 serves to definethe outer boundaries of counter electrodes 434A and 434B and define theinterior walls of well 430. In another embodiment, boundary 440 does notextend beneath counter electrodes 434A and 434B.

FIG. 4C illustrates well 460, another embodiment of well 158. Well 460comprises wall 461 having an interior surface 462, counter electrodes464A and 464B and working electrode 474. Well 460 may, optionally,comprise a reference electrode (not shown). The exposed region ofworking electrode 474 is defined by boundary 470. Boundary 470 may abutcounter electrodes 464A and 464B and may abut interior surface 462.Alternatively, boundary 470 may extend under or over counter electrodes464A and 464B. In some embodiments, boundary 470 extends beneath counterelectrodes 464A and 464B and electrically isolates them from workingelectrode 474. Boundary 470 may be comprised of a material deposited onworking electrode 474 and may or may not be comprised of the samematerial as working electrode 474. Boundary 470 may be a region in whichmaterial has been removed from working electrode 474. Boundary 470 mayalso comprise a region of working electrode 474 with different physical,chemical or compositional properties. Preferably, boundary 470 iscomposed of a dielectric material deposited on the surface of workingelectrode 474. Boundary 470 may be coplanar with working electrode 474and may extend out from or into the surface of working electrode 474.Working electrode 474 may have assay domains as described above. Inpreferred embodiments, boundary 470 confines materials (e.g. liquids,assay reagents, and the like) on working electrode 474. Boundary 470 mayalso be used to aid in or direct the deposition of materials to workingelectrode 474, for example, by preventing spreading of liquids tosurrounding regions of working electrode 470 or to counter electrodes464A and 464B. Boundary 470 may also serve as indicia duringmeasurements using the apparatus of the present invention. In apreferred embodiment: working electrode 474 is a conducting material,either self supporting or supported on another material; boundary 470 isa non-conducting material deposited on working electrode 474 that coversworking electrode 474; counter electrodes 464A and 464B are deposited onboundary 470 and are electrically isolated from working electrode 474 byboundary 470; and wall 461 with interior surface 462 serves to definethe outer boundaries of counter electrodes 464A and 464B and define theinterior walls of well 460. In another embodiment, boundary 470 isdeposited on working electrode 474 so that it does not extend over orunder counter electrodes 464A and 464B. Well 460 has working electrode474 with assay domains 476, as described above for wells 300, 330 and360. In another embodiment, assay domains 476 on working electrode 474are defined by additional boundaries as described above for well 400.

FIG. 4D illustrates well 480, another embodiment of well 158. Well 480comprises wall 482 with interior surface 484, counter electrodes 488Aand 488B, boundary 492 and working electrode 494. Well 480 may,optionally, comprise a reference electrode (not shown). Regions 499A and499B of working electrode 494 are defined by boundaries 498A and 498B.Boundaries 498A and 498B may be comprised of a material deposited onworking electrode 494 or may be comprised on the same material asworking electrode 494. Boundaries 498A and 498B may be regions in whichmaterial has been removed from working electrode 494. Boundaries 498Aand 498B may also comprise regions of working electrode 494 withdifferent physical, chemical or compositional properties. For example,boundaries 498A and 498B may be hydrophilic or hydrophobic, have high orlow surface area, and/or an area of high or low conductivity.Preferably, boundaries 498A and 498B are composed of non-conducting ordielectric materials deposited on the surface of working electrode 494and may provide a physical boundary. Boundaries 498A and 498B may becoplanar with working electrode 494 and may extend out from or into thesurface of working electrode 494. Exposed working electrode regions 499Aand 499B may comprise assay domains as described above. In preferredembodiments, boundaries 498A and 498B confine materials (e.g. liquids,assay reagents, and the like) on working electrode 494. Boundaries 498Aand 498B may also be used to aid in or direct the deposition ofmaterials to interior regions 499A and 499B, for example, by preventingspreading of liquids deposited to surrounding regions of workingelectrode 494 or to counter electrodes 488A and 488B. Boundaries 498Aand 498B may also serve as an indicia during measurements using theapparatus of the present invention.

FIG. 4E illustrates well 4900, another embodiment of well 158. Well 4900comprises wall 4902 with interior surface 4903, counter electrodes 4904Aand 4904B, gaps 4906A and 4906B (the gaps preferably being dielectricsurfaces separating working electrode 4910 from counter electrodes 4904Aand 4904B) and barrier 4908 with a plurality of holes 4912 that exposeworking electrode 4910. Well 4900 may, optionally, comprise a referenceelectrode (not shown). In a preferred embodiment, boundary 4908 may be adielectric material that provides a boundary that can confine smallvolumes of fluid to the exposed regions of the electrode (e.g., so as toallow the controlled immobilization of reagents onto defined assaydomains on working electrode 4910). Working electrode 4910 may haveassay reagents immobilized on its surface in regions where plurality ofholes 4912 in boundary 4908 expose working electrode 4910. Boundary 4908may also be used to aid in or direct the deposition of materials toworking electrode 4910 where holes 4912 expose working electrode 4910.

5.1.2 Electrodes

One aspect of the invention relates to improved electrode compositionsand surfaces and assay modules comprising these electrode compositionsand surfaces. Electrodes in the present invention are preferablycomprised of a conductive material. The electrode may comprise a metalsuch as gold, silver, platinum, nickel, steel, iridium, copper,aluminum, a conductive alloy, or the like. They may also comprise oxidecoated metals (e.g. aluminum oxide coated aluminum). According to oneembodiment, the working and counter electrodes are not the same material(e.g. metal counter electrode and carbon working electrode). Preferably,electrodes are comprised of carbon-based materials such as carbon,carbon black, graphitic carbon, carbon nanotubes, carbon fibrils,graphite, carbon fibers and mixtures thereof. Preferably, the electrodescomprise elemental carbon (e.g., graphitic, carbon black, carbonnanotubes, etc.). Advantageously, they may be comprised of conductingcarbon-polymer composites, conducting particles dispersed in a matrix(e.g. carbon inks, carbon pastes, metal inks), and/or conductingpolymers. One preferred embodiment of the invention is an assay module,preferably a multi-well plate, having electrodes (e.g., working and/orcounter electrodes) that comprise carbon, preferably carbon layers, morepreferably screen-printed layers of carbon inks. Some useful carbon inksinclude materials produced by Acheson Colloids Co. (e.g., Acheson 440B,423ss, PF407A, PF407C, PM-003A, 30D071, 435A, Electrodag 50SSS, andAquadag™), E. I. Du Pont de Nemours and Co. (e.g., Dupont 7105, 7101,7102, 7103, 7144, 7082, 7861D, and CB050), Conductive Compounds Inc(e.g., C-100), and Ercon Inc. (e.g., G-451).

Electrodes may also be comprised of semiconducting materials (e.g.silicon, germanium) or semi-conducting films such as indium tin oxide(ITO), antimony tin oxide (ATO) and the like. Electrodes may also becomprised of mixtures of materials containing conducting composites,inks, pastes, polymer blends, metal/non-metal composites and the like.Such mixtures may include conductive or semi-conductive materials mixedwith non-conductive materials. Preferably, electrode materials aresubstantially free of silicone-based materials. Electrodes may be formedinto patterns by a molding process (i.e., during fabrication of theelectrodes), by patterned deposition, by patterned printing, byselective etching, through a cutting process such as die cutting orlaser drilling, and/or by techniques known in the art of electronicsmicrofabrication (e.g., chemical etching, photopatterning of a resistmaterial, microlithographic techniques, etc.).

The terms “carbon fibrils”, “carbon nanotubes”, single wall nanotubes(SWNT), multiwall nanotubes (MWNT), “graphitic nanotubes”, “graphiticfibrils”, “carbon tubules”, “fibrils” and “buckeytubes”, all of whichterms may be used to describe a broad class of carbon materials (seeDresselhaus, M. S.; Dresselhaus, G.; Eklund, P. C.; “Science ofFullerenes and Carbon Nanotubes”, Academic Press, San Diego, Calif.,1996, and references cited therein). The terms “fibrils” and “carbonfibrils” are used throughout this application to include this broadclass of carbon-based materials.

Individual carbon fibrils as disclosed in U.S. Pat. Nos. 4,663,230;5,165,909; and 5,171,560 are particularly advantageous. They may havediameters that range from about 3.5 nm to 70 nm, and length greater than10 times the diameter, an outer region of multiple, essentiallycontinuous, layers of ordered carbon atoms and a distinct inner coreregion. Simply for illustrative purposes, a typical diameter for acarbon fibril may be approximately between about 7 and 25 nm, and atypical range of lengths may be 1000 nm to 10,000 nm. Carbon fibrils mayalso have a single layer of carbon atoms and diameters in the range of 1nm-2 nm.

Carbon materials can be made to form aggregates. For example, asdisclosed in U.S. Pat. No. 5,110,693, and references cited therein, twoor more individual carbon fibrils may form microscopic aggregates ofentangled fibrils. These aggregates can have dimensions ranging from 5nm to several cm. Simply for illustrative purposes, one type ofmicroscopic aggregate (“cotton candy or CC”) resembles a spindle or rodof entangled fibers with a diameter that may range from 5 nm to 20,000nm with a length that may range from 100 nm to 1 mm. Again forillustrative purposes, another type of microscopic aggregate of fibrils(“birds nest, or BN”) can be roughly spherical with a diameter that mayrange from 0.1 urn to 1000 um. Larger aggregates of each type (CC and/orBN) or mixtures of each can be formed (vide infra).

Fibrils that can be used in the present invention include but are notlimited to individual fibrils, aggregates of one or more fibrils,suspensions of one or more fibrils, dispersions of fibrils, mixtures offibrils with other materials (e.g., oils, paraffins, waxes, polymers,gels, plastics, adhesives, epoxies, teflon, metals, organic liquids,organic solids, inorganic solids, acids, bases, ceramics, glasses,rubbers, elastomers, biological molecules and media, etc.) as well ascombinations thereof. One preferred embodiment of the invention relatesto a multi-well plate comprising a substrate comprising a carbonnanotube-containing composite, wherein the surface of the substrate isetched to expose the carbon nanotubes, thereby forming one or moreworking electrodes.

Electrodes may be self supporting or may be supported on anothermaterial, e.g. on films, plastic sheets, adhesive films, paper,backings, meshes, felts, fibrous materials, gels, solids (e.g. metals,ceramics, glasses), elastomers, liquids, tapes, adhesives, otherelectrodes, dielectric materials and the like. The support may be rigidor flexible, flat or deformed, transparent, translucent, opaque orreflective. Preferably, the support comprises a flat sheet of plasticsuch as acetate, polycarbonate, polypropylene, polyester (e.g., Mylar),polyimide (e.g., Kapton), or polystyrene. According to one embodiment,the material comprises polystyrene blended with High Impact Polystyrene(HIPS) to reduce the brittleness of the material. Preferably, between 4and 16 wt % HIPS is blended with the polystyrene, more preferablybetween about 8 and 12 wt %. Electrode materials may be applied to asupport by a variety of coating and deposition processes known in theart such as painting, spray-coating, screen-printing, ink-jet printing,laser printing, spin-coating, evaporative coating, chemical vapordeposition, laminating, etc. Supported electrodes may be patterned usingphotolithographic techniques (e.g., established techniques in themicrofabrication of electronics), by selective etching, and/or byselective deposition (e.g., by evaporative or CVD processes carried outthrough a mask). In a preferred embodiment, electrodes are comprised ofextruded films of conducting carbon/polymer composites. In anotherpreferred embodiment, electrodes are comprised of a screen printedconducting ink deposited on a substrate. Yet another embodiment involvesthe combination of a counterelectrode comprising a chemically etchedmetal (e.g., steel) or die-cut aluminized film and a screen-printedworking electrode.

Electrodes may be supported by another conducting material.Advantageously, conducting carbon electrodes may be in contact withconducting metal pastes. Preferably, electrodes are (or are capable ofbeing) derivatized or modified, for example, to immobilize assayreagents such as binding reagents on electrodes. One may attach, e.g.,antibodies, fragments of antibodies, proteins, enzymes, enzymesubstrates, inhibitors, cofactors, antigens, haptens, lipoproteins,liposaccharides, bacteria, cells, sub-cellular components, cellreceptors, viruses, nucleic acids, antigens, lipids, glycoproteins,carbohydrates, peptides, amino acids, hormones, protein-binding ligands,pharmacological agents, and/or combinations thereof. It may also bedesirable to attach non-biological entities such as, but not limited topolymers, elastomers, gels, coatings, ECL tags, redox active species(e.g., tripropylamine, oxalates), inorganic materials, chemicalfunctional groups, chelating agents, linkers etc. Reagents may beimmobilized on the electrodes by a variety of methods including passiveadsorption, specific binding and/or through the formation of covalentbonds to functional groups present on the surface of the electrode.

Electrodes may be modified by chemical or mechanical treatment toimprove the immobilization of reagents. The surface may be treated tointroduce functional groups for immobilization of reagents or to enhanceits adsorptive properties. Surface treatment may also be used toinfluence properties of the electrode surface, e.g., the spreading ofwater on the surface or the kinetics of electrochemical processes at thesurface of the electrode. Techniques that may be used include exposureto electromagnetic radiation, ionizing radiation, plasmas or chemicalreagents such as oxidizing agents, electrophiles, nucleophiles, reducingagents, strong acids, strong bases and/or combinations thereof.Treatments that etch one or more components of the electrodes may beparticularly beneficial by increasing the roughness and therefore thesurface area of the electrodes. In the case of composite electrodeshaving conductive particles or fibers (e.g., carbon particles orfibrils) in a polymeric matrix or binder, selective etching of thepolymer may be used to expose the conductive particles or fibers.

One particularly useful embodiment is the modification of the electrode,and more broadly a material incorporated into the present invention bytreatment with a plasma, specifically a low temperature plasma, alsotermed glow-discharge. The treatment is carried out in order to alterthe surface characteristics of the electrode, which come in contact withthe plasma during treatment. Plasma treatment may change, for example,the physical properties, chemical composition, or surface-chemicalproperties of the electrode. These changes may, for example, aid in theimmobilization of reagents, reduce contaminants, improve adhesion toother materials, alter the wettability of the surface, facilitatedeposition of materials, create patterns, and/or improve uniformity.Examples of useful plasmas include oxygen, nitrogen, argon, ammonia,hydrogen, fluorocarbons, water and combinations thereof. Oxygen plasmasare especially preferred for exposing carbon particles in carbon-polymercomposite materials. Oxygen plasmas may also be used to introducecarboxylic acids or other oxidized carbon functionality into carbon ororganic materials (these may be activated, e.g., as active esters oracyl chlorides) so as to allow for the coupling of reagents. Similarly,ammonia-containing plasmas may be used to introduce amino groups for usein coupling to assay reagents.

Treatment of electrode surfaces may be advantageous so as to improve orfacilitate reagent immobilization, change the wetting properties of theelectrode, increase surface area, increase the binding capacity for theimmobilization of reagents or the binding of analytes, and/or alter thekinetics of electrochemical reactions at the electrode. In someapplications, however, it may be preferable to use untreated electrodes.For example, we have found that it is advantageous to etch carbon inkelectrodes prior to adsorbing binding reagents (e.g., avidin,streptavidin or antibodies) when the application calls for a largedynamic range and therefore a high binding capacity per area ofelectrode. We have discovered that oxidative etching (e.g., by oxygenplasma) has additional advantages in that the potential for oxidation oftripropyl amine (TPA) and the contact angle for water are both reducedrelative to the unetched ink. The low contact angle for water allowsreagents to be adsorbed on the electrode by application of the reagentsin a small volume of aqueous buffer and allowing the small volume tospread evenly over the electrode surface. Surprisingly, we have foundthat excellent assays may also be carried out on unetched carbon inkelectrodes despite the presence of polymeric binders in the ink. Infact, in some applications requiring high sensitivity or low-nonspecific binding it is preferred to use unetched carbon ink electrodesso as to minimize the surface area of exposed carbon and thereforeminimize background signals and loss of reagents from non-specificbinding of reagents to the exposed carbon. Depending on the ink used andthe process used to apply the ink, the electrode surface may not beeasily wettable by aqueous solutions. We have found that we cancompensate for the low wettability of the electrodes during theadsorption of reagents by adding low concentrations of non-ionicdetergents to the reagent solutions so as to facilitate the spreading ofthe solutions over the electrode surface. Even spreading is especiallyimportant during the localized immobilization of a reagent from a smallvolume of solution. For example, we have found that the addition of0.005-0.04% Triton X-100® allows for the spreading of protein solutionsover unetched carbon ink surfaces without affecting the adsorption ofthe protein to the electrode and without disrupting the ability of adielectric film applied on or adjacent to the electrode (preferably, aprinted dielectric film with a thickness of 0.5-100 micrometers, or morepreferably 2-30 micrometers, or most preferably 8-12 micrometers andhaving a sharply defined edge) to confine fluids to the electrodesurface. Preferably, when non-ionic detergents such as Triton X-100 areused to facilitate spreading of capture reagents onto unetchedscreen-printed electrodes (i.e., so as to allow the immobilization ofthe capture reagents), the solutions containing the capture reagents areallowed to dry onto the electrode surface. It has been found that thisdrying step greatly improves the efficiency and reproducibility of theimmobilization process.

Electrodes can be derivatized with chemical functional groups that canbe used to attach other materials to them. Materials may be attachedcovalently to these functional groups, or they may be adsorbednon-covalently to derivatized or underivatized electrodes.

Electrodes may be prepared with chemical functional groups attachedcovalently to their surface. These chemical functional groups includebut are not limited to COOH, OH, NH₂, activated carboxyls (e.g.,N-hydroxy succinimide (NHS)-esters), poly-(ethylene glycols), thiols,alkyl ((CH₂)_(n)) groups, and/or combinations thereof). Certain chemicalfunctional groups (e.g., COOH, OH, NH₂, SH, activated carboxyls) may beused to couple reagents to electrodes. For further reference to usefulimmobilization and bioconjugation techniques see G. Hermanson, A. Malliaand P. Smith, Immobilized Affinity Ligand Techniques (Academic Press,San Diego, 1992) and G. Hermanson, Bioconjugate Techniques (AcademicPress, San Diego, 1996).

In preferred embodiments, NHS-ester groups are used to attach othermolecules or materials bearing a nucleophilic chemical functional group(e.g., an amine). In a preferred embodiment, the nucleophilic chemicalfunctional group is present on and/or in a biomolecule, either naturallyand/or by chemical derivatization. Examples of suitable biomoleculesinclude, but are not limited to, amino acids, proteins and functionalfragments thereof, antibodies, binding fragments of antibodies, enzymes,nucleic acids, and combinations thereof. This is one of many suchpossible techniques and is generally applicable to the examples givenhere and many other analogous materials and/or biomolecules. In apreferred embodiment, reagents that may be used for ECL may be attachedto the electrode via NHS-ester groups.

A reagent that can be used in an ECL assay can be attached to electrodesby covalent bonds (e.g., reaction with an NHS-ester), by reaction withan appropriate linker (vide supra), by non-specific binding, and/or by acombination thereof.

It may be desirable to control the extent of non-specific binding ofmaterials to electrodes. Simply by way of non-limiting examples, it maybe desirable to reduce or prevent the non-specific adsorption ofproteins, antibodies, fragments of antibodies, cells, subcellularparticles, viruses, serum and/or one or more of its components, ECLlabels (e.g., Ru^(II)(bpy)₃ and Ru^(III)(bpy)₃ derivatives), oxalates,trialkylamines, antigens, analytes, and/or combinations thereof). Inanother example, it may be desirable to enhance the binding ofbiomolecules.

One or more chemical moieties that reduce or prevent non-specificbinding (also known as blocking groups) may be present in, on, or inproximity to an electrode. Such moieties, e.g., PEG moieties and/orcharged residues (e.g., phosphates, ammonium ions), may be attached toor coated on the electrode. Examples of useful blocking reagents includeproteins (e.g., serum albumins and immunoglobins), nucleic acids,polyethylene oxides, polypropylene oxides, block copolymers ofpolyethylene oxide and polypropylene oxide, polyethylene imines anddetergents or surfactants (e.g., classes of non-ionicdetergents/surfactants known by the trade names of Brij, Triton, Tween,Thesit, Lubrol, Genapol, Pluronic, Tetronic, and Span).

Materials used in electrodes may be treated with surfactants to reducenon-specific binding. For example, electrodes may be treated withsurfactants and/or detergents that are well known to one of ordinaryskill in the art (for example, the Tween series, Triton, Span, Brij).Solutions of PEGs and/or molecules which behave in similar fashion toPEG (e.g., oligo- or polysaccharides, other hydrophilic oligomers orpolymers) (“Polyethylene glycol chemistry. Biotechnical and BiomedicalApplications”, Harris, J. M. Editor, 1992, Plenum Press) may be usedinstead of and/or in conjunction with surfactants and/or detergents.Undesirable non-specific adsorption of certain entities such as thoselisted above may be blocked by competitive non-specific adsorption of ablocking agent, e.g., by a protein such as bovine serum albumin (BSA) orimmunoglobulin G (IgG). One may adsorb or covalently attach an assayreagent on an electrode and subsequently treat the electrode with ablocking agent so as to block remaining unoccupied sites on the surface.

In preferred embodiments, it may be desirable to immobilize (by eithercovalent or non-covalent means) biomolecules or other media tocarbon-containing materials, e.g., carbon black, fibrils, and/or carbondispersed in another material. One may attach antibodies, fragments ofantibodies, proteins, enzymes, enzyme substrates, inhibitors, cofactors,antigens, haptens, lipoproteins, liposaccharides, cells, sub-cellularcomponents (e.g., organelles or membrane fragments), cell receptors,viruses, nucleic acids, antigens, lipids, glycoproteins, carbohydrates,peptides, amino acids, hormones, protein-binding ligands,pharmacological agents, and/or combinations thereof. It may also bedesirable to attach non-biological entities such as, but not limited topolymers, elastomers, gels, coatings, ECL tags, redox active species(e.g., tripropylamine, oxalates), inorganic materials, chelating agents,linkers etc. A plurality of species may be co-adsorbed to form a mixedlayer on the surface of an electrode.

Electrodes used in the multi-well assay plates of the invention aretypically non-porous, however, in some applications it is advantageousto use porous electrodes (e.g., mats of carbon fibers or fibrils,sintered metals, and metals films deposited on filtration membranes,papers or other porous substrates. These applications include those thatemploy filtration of solutions through the electrode so as to: i)increase mass transport to the electrode surface (e.g., to increase thekinetics of binding of molecules in solution to molecules on theelectrode surface); ii) capture particles on the electrode surface;and/or iii) remove liquid from the well.

Electrodes used in assay modules of the invention are advantageouslyable to induce luminescence from luminescent species. It is preferablethat electrodes are comprised of materials that are compatible withbiological media, impervious to the reagents typically encountered inluminescence measurements, and robust.

A working electrode may have one or more of the properties describedabove generally for electrodes. Preferably materials for workingelectrodes are materials able to induce electrochemiluminescence fromRuthenium-tris-bipyridine in the presence of tertiary alkyl amines (suchas tripropyl amine). Examples of such preferred materials includeplatinum, gold, ITO, carbon, carbon-polymer composites, and conductivepolymers. In one embodiment, the working electrode is made of acontinuous conducting sheet or a film of one or more conductingmaterials. This sheet or film may be extruded, pressed or molded, andcan be self supporting. In a preferred embodiment, the working electrodeis made of a carbon-polymer composite. The composite may be comprised ofconducting carbon particles (e.g., carbon fibrils, carbon black,graphitic carbon) dispersed in a matrix (e.g., a polymer such as EVA,polystyrene, polyethylene, ABS). The working electrode may additionallycomprise other conducting materials, for example, a conducting metal inkmay be printed on the conducting composite.

In another embodiment, the working electrode is made of a conductingmaterial deposited and/or patterned on a substrate (e.g., by printing,painting, coating, spin-coating, evaporation, chemical vapor deposition,electrolytic deposition, electroless deposition, photolithography andother electronics microfabrication techniques, etc.). In a preferredembodiment, the working electrode comprises a conductive carbon inkprinted on a polymeric support (e.g., by ink-jet printing, laserprinting, or, most preferably, by screen-printing). The workingelectrode may be a continuous film, it may be one or more discreteregions (e.g., patterns), or it may be a plurality of connected regions:The working electrode may additionally comprise other conductingmaterials, for example, a carbon ink overlayer may be deposited over aconducting metal ink (e.g., a silver ink) underlayer, the underlayerbeing used to increase the conductivity of the film. It may bebeneficial to print or deposit the overlayer in multiple layers so as toensure that the underlayer is completely covered so that the underlayerdoesn't interfere with subsequent processing steps or with ECLmeasurements (e.g., a preferred electrode material comprises two layers,preferably three layers, of carbon ink over a layer of silver ink, thelayers most preferably being deposited by screen printing).Alternatively, one or two layers of carbon may be used. For electrodescomprising one or more printed carbon ink layers over a printed silverink layer, the silver layer has a thickness of, preferably, 2.5 micronsto 25 microns, more preferably, 4-7 microns (or, alternatively, athickness that produces a resistance of, preferably less than 2ohms/square or, more preferably, 0.05-0.2 ohms/square) and the combinedcarbon layers have a thickness of, preferably, 2.5-75 microns or, morepreferably, 6-25 microns (or, alternatively, a thickness that produces aresistance of, preferably less than 100 ohms/square or, more preferably,less than 30 ohms/square or, most preferably 20-30 ohms/square).

A counter electrode may have one or more of the properties describedabove generally for electrodes and for working electrodes. In oneembodiment, the counter electrode is made of a continuous conductingsheet or a film of one or more conducting materials. This sheet or filmmay be extruded, pressed or molded, and can be self supporting. In apreferred embodiment, the counter electrode is made of a carbon-polymercomposite. The composite may be comprised of conducting carbon particles(e.g., carbon fibrils, carbon black, graphitic carbon) dispersed in amatrix (e.g., a polymer such as EVA, polystyrene, polyethylene, ABS).The counter electrode may additionally comprise other conductingmaterials, for example, a conducting metal ink may be printed on theconducting composite.

In another embodiment, the counter electrode comprises a metal coating,film or foil. One preferred embodiment of the invention is a multi-wellplate having wells containing (preferably in two or more wells of theplate) working electrodes that comprise carbon (preferably carbon ink orcarbon particles, e.g., carbon nanotubes, dispersed in a matrix) andcounter electrodes comprising a metal coating, film or sheet or foil(preferably, comprising aluminum, stainless steel, nickel or silver). Afoil counterelectrode may be self-supporting or may be supported onanother material. It may also additionally comprise an adhesivematerial, a non-conducting layer and/or a backing material. The foil mayhave holes, advantageously in a pattern that corresponds to the patternof wells in industry standard multi-well assay plates. Holes may bepunched, drilled, burned, laser drilled, machined, etched or otherwiseintroduced by removing material from a continuous film, or, the filmmany be generated (e.g., molded) to incorporate holes. In a preferredembodiment, the counter electrode is formed from a plastic sheet orsupport that is coated on one side with an aluminum film or foil andcoated on the opposite side with an adhesive layer, preferably, having aremovable backing strip.

In another embodiment, the counter electrode is made of a conductingmaterial deposited and/or patterned on a substrate (as described abovefor the working electrode). In a preferred embodiment, the counterelectrode comprises a conducting carbon ink printed on a polymericsupport. The counter electrode may be a continuous film, it may be oneor more discrete regions (e.g., patterns), or it may be a plurality ofconnected regions. The counter electrode may additionally comprise otherconducting materials, for example, a conducting metal ink (e.g., asilver ink) may be printed on the substrate and may be in contact withthe conducting ink of the counter electrode.

An unexpected feature of the instrumentation of the invention is itsability to conduct precise, accurate and reproducible electrode inducedluminescent assays, particularly electrochemiluminescent assays, withoutthe use of an independent reference electrode and potentiostat (i.e.,without using a three electrode configuration: working, counter andreference). In a two electrode system (working and counter electrode)any potential applied across the working and counter electrodes isdistributed, at least in part, over the two electrode/solutioninterfaces. The undefined nature of the potential at the surface of thecounter electrode leads directly to uncertainty in the potential at theworking electrode. This problem may be solved by using a counterelectrode with a stable interfacial potential that is defined by a redoxcouple in solution or, preferably, by a redox couple confined to thesurface of the electrode (such a counter electrode is sometimes termed a“counter/reference electrode”). Some examples of useful“counter/reference electrode” materials include metal/metal halidecouples such as silver/silver chloride; metal/metal oxide couples suchas silver/silver oxide, nickel/nickel oxide and zinc/zinc oxide; andmetal oxides with allowing for multiple metal oxidation states such asmanganese oxide. For optimal performance, these “counter/referenceelectrodes” should have a sufficiently high concentration of accessibleredox species so as to prevent polarization of the electrode during thecourse of an ECL measurement.

Surprisingly, we have observed excellent performance and precision(e.g., coefficients of variation of <10%, more preferably <5%, even morepreferably 2% and most preferably <1%) in ECL measurements using twoelectrode configurations and counter electrodes that are not typicallyconsidered useful “counter/reference electrodes”, for example: aluminum(presumably with a native oxide layer) and various forms of carbon(including composites containing carbon black, graphite aid/or carbonfibrils). Without being bound by theory, we believe this unanticipatedperformance has been achieved by i) maintaining a consistent andreproducible process for the manufacture of ECL multi-well assay plates;ii) maintaining a relatively consistent chemical environment duringinduction of ECL and/or iii) selection of appropriate voltage or currentwaveforms. In general, under the high current conditions typically usedto generate ECL, the interfacial potential at the counter electrode isdetermined by the reduction potential for water at that electrode; aslong as the electrode surface and chemical environment remain relativelyconsistent the interfacial potential can be highly reproducible. Thevoltage/current waveforms used to induce ECL, preferably, involve the i)application of voltage or current sufficient to induce ECL and ii) themaintenance of ECL until the ECL intensity decays (presumably due toconsumption of ECL coreactant or destruction of assay components on theelectrode surface). Under these conditions, a plot of ECL vs. time hasthe form of a peak. Such waveforms are tolerant of some inconsistency inworking electrode and counter electrode potential, solution resistanceand the like; these variations tend to shift the start and end of theECL peak but have a much smaller effect on the total integrated lightsignal under the peak. An especially preferred voltage/current waveformis a voltage ramp beginning at a voltage less than that required toinduce ECL and ending at a potential high enough to allow decay of theECL signal to under 10% of the peak intensity.

Surprisingly, we have also found that we can attain excellentperformance and precision (e.g., coefficients of variation of <10%, morepreferably <5%, even more preferably 2% and most preferably <1%) in ECLmeasurements using two electrode systems despite using counterelectrodes having exposed geometric surface areas that are equal or lessthan the exposed geometric surface area of the working electrode. Suchexcellent performance is attained even when using the unconventionalcounter electrode materials described above. By contrast, in standardelectrochemical assays it is considered highly advantageous to havelarger counter electrodes than working electrodes to ensure that thecurrent is not limited by chemical or mass transport processes at thecounter electrode. Reducing the surface area of the counter electrodegives certain advantages in the design of multi-well assay plates forECL assays; by reducing the counter electrode area it is possible toincrease the area of the active working electrode and thereby thekinetics of reactions occurring at the surface of the electrode, thebinding capacity of assays using binding reagents immobilized on theworking electrode and/or the number of assay domains that may bepatterned on a given working electrode. In preferred embodiments of themulti-well plates of the invention, the ratio of the geometric surfaceareas of the working and counter electrodes is greater than 1, greaterthan 2, greater than 5, greater than 10, greater than 50 or, mostpreferably, greater than 100.

While in many applications it is advantageous to have an electrodesurface area that occupies a large fraction of an assay region (forreasons described above), in other applications it may be advantageousto have small exposed working electrode surfaces (preferably less than 4mm², more preferably less than 1 mm², even more preferably less than 0.1mm² and most preferably less than 0.01 mm²). For example, small workingelectrode surfaces may in some cases lead to higher sensitivity andlower non-specific signals. For example, in a binding assay conductedusing a binding reagent immobilized on the working electrode, the signalfrom a labeled binding partner of the binding reagent should be roughlyindependent of the area of the electrode (assuming the binding capacityof the electrode is sufficient to bind all the labeled binding partnerand the binding reaction is allowed to proceed to completion).Non-specific signals, e.g., due to non-specific binding, should beroughly linearly dependent on electrode area. Under such conditions,reducing electrode area may lead to an improvement in the ratio ofspecific to non-specific signal. According to one embodiment ofmulti-well assay plates of the invention, the well bottoms compriseworking electrodes and the ratio of working electrode surface to thesurface area of the bottom of the well (or, alternatively, to dielectricsurfaces on the bottom of the well) is less than 1 to 5, preferably 1 to10, more preferably 1 to 30.

Despite the excellent performance we have observed with two electrodesystems, some specialized applications may require multi-well assayplates having independent reference electrodes so as to allow control ofthe working electrode potentials with a potentiostat. Referenceelectrodes may be made using the materials and methods described abovefor working and counter electrodes. Preferably the reference electrodehas a stable interfacial potential that is defined by a redox coupleconfined to the surface of the electrode; examples of materials havingthis property include metal/metal halide couples such as silver/silverchloride; metal/metal oxide couples such as silver/silver oxide,nickel/nickel oxide and zinc/zinc oxide; and metal oxides with allowingfor multiple metal oxidation states such as manganese oxide. Manyreference electrode materials have surface potentials that are dependenton their chemical environment (e.g., on the pH or concentration ofhalide ions). If necessary, reference electrodes may be protected fromvariations in the chemical environment by coating the electrode with afilm (e.g., a hydrophilic polymeric film) that provides for a welldefined chemical environment (e.g., controlled concentrations ofhydrogen ions or halide ions) directly on the surface of the electrodebut also allows for the passage of ions in and out of the film. It maybe advantageous to cover a substantial portion of these polymer filmswith an ion impermeable film so as to balance the requirement for ionflow in and out of the polymer film with the requirement that thechemical environment in the film remain substantially unaffected bycontact with a sample or reagent solution (see, e.g., U.S. Pat. Nos.5,384,031 and 4,933,048).

The electrodes and power sources of the invention may be directlyconnected or may be connected via a conductive lead or pathway,preferably formed of a conductive matrix such as a metal, a conductivecarbon-containing material or composite, a conductive polymer or anelectrolytic solution. One embodiment of the invention relates to assaymodules comprising electrodes connected to electrical power sources viaelectrolytic solutions (e.g., so called “floating electrodes”), suchelectrodes being, preferably, adapted for inducing electrode inducedluminescence (most preferably, electrochemiluminescence). By way ofexample, in one embodiment of the well of a multi-well assay platepictured in FIG. 4E, counter electrodes 4904A and 4904B may be adaptedto be independently connected to the two poles of a source of electricalenergy. In the use of this embodiment, the application of a potentialacross electrodes 4904A and 4904B is used to induce an electricalpotential in electrode 4910 and, preferably, to induce luminescence(most preferably, electrochemiluminescence) from luminescent labels atelectrode 4910.

5.1.3 Dielectrics

The assay modules of the present invention may use dielectric inks,films or other electrically insulating materials (hereinafter referredto as dielectrics). Dielectrics in the present invention may be used toprevent electrical connectivity between electrodes, to define patternedregions, to adhere materials together (i.e., as adhesives), to supportmaterials, to define assay domains, as masks, as indicia and/or tocontain assay reagents and other fluids. Dielectrics are non-conductingand advantageously non-porous (i.e., do not permit transmission ofmaterials) and resistant to dissolving or degrading in the presence ofmedia encountered in an electrode induced luminescence measurement. Thedielectrics in the present invention may be liquids, gels, solids ormaterials dispersed in a matrix. They may be deposited in uncured formand cured to become solid. They may be inks, solid films, tapes orsheets. Materials used for dielectrics include polymers, photoresists,solder masks, plastics, adhesives, gels, glasses, non-conducting inks,non-conducting pastes, ceramics, papers, elastomers, silicones,thermoplastics. Preferably, dielectric materials of the invention aresubstantially free of silicones. Examples of non-conducting inks includeUV curable dielectrics such as materials produced by Acheson ColloidsCo. (e.g., Acheson 451SS, 452SS, PF-021, PD-039, ML25251, ML25240,ML25265, and Electrodag 38DJB16 clear), Nazdar Inc. (SPL 4000 series ofhalf tone inks) and E. I. du Pont de Nemours and Co. (e.g., Dupont:5018, 3571, and 5017).

Dielectrics of the present invention may be applied by a variety ofmeans, for example, printing, spraying, laminating, or may be affixedwith adhesives, glues, solvents or by use of mechanical fasteners.Patterns and/or holes in dielectric layers may be formed by moldingprocesses (i.e., during fabrication of the layer), by selective etchingand/or by a cutting process such as die cutting or laser drilling.Dielectrics may be deposited and/or etched in patterns through the useof established photolithographic techniques (e.g., techniques used inthe semiconductor electronics industry) and/or by patterned depositionusing an evaporative or CVD process (e.g., by deposition through amask). In a preferred embodiment, a dielectric ink is deposited on asubstrate by printing (e.g., ink jet printing, laser printing or, morepreferably, screen printing) and, optionally, UV cured. Preferably, thescreen printed dielectric is UV curable allowing for improved edgedefinition than solvent based dielectrics. In another preferredembodiment, a non-conducting polymeric film is affixed to a supportusing an adhesive.

When using a dielectric ink printed on or adjacent an electrode toconfine fluids to regions of the electrode surface, the dielectric filmpreferably has a thickness of 0.5-100 micrometers, or more preferably2-30 micrometers, or most preferably 8-12 micrometers and also,preferably, has a sharply defined edge with steep walls.

5.1.4 Plate Tops

The invention includes plate tops and assembled plates comprising aplate top and, preferably, a plate bottom defining well bottoms havingone or more electrode surfaces, most preferably having one or moreworking electrode surfaces and, optionally, one or more counterelectrode surfaces. Preferably, the plate top is a structure with holes,wherein the structure may be combined with a plate bottom to form amulti-well plate, the walls of the wells of the plate being at leastpartially defined by the inside surfaces of the holes through the platetop. The holes through the plate top may be a variety of shapes (e.g.,round, oval, square, rectangular, triangular, star shaped, etc.). Theholes may be of various sizes. They can also have irregular dimensionswithin a hole (e.g., the hole may become more narrow or more wide atdifferent depths). For example, the hole may be shaped like a cone,becoming more narrow at the bottom so as to optimize the collection oflight emitted from the well bottom. The plate top may also havestructures or indicia thereon that aid in identifying the plate top,distinguishing the plate top from other configurations of plate top, orin aligning and handling the plate top. Advantageously, the dimensionsand structure of the plate top are preferably in accordance with, or atleast compatible with, industry standards for the footprints and shapesof assay plates.

The plate top may be made from conducting or non-conducting materials.Preferably, the majority of the plate top is a unitary molded structuremade from rigid thermoplastic material such as polyethylene, acetate,polycarbonate, polypropylene, polyester (e.g., Mylar), polyimide (e.g.,Kapton), or polystyrene. Preferably, the support comprises a flat sheetof plastic. According to one embodiment, the material comprisespolystyrene blended with High Impact Polystyrene (HIPS) to reduce thebrittleness of the material. Preferably, between 4 and 16 wt % HIPS isblended with the polystyrene, more preferably between about 8 and 12 wt%. Optimally, this unitary structure is formed of (or, alternatively,coated with) inexpensive material that is generally impervious toreactants, can withstand modest levels of heat and light and is,preferably, resistant to the adsorption of biomolecules. Preferably, theplate top is substantially free of silicones. Plate tops may be clear ortranslucent. Different colored materials may be used to improve theresults of certain ECL measurement processes.

It is preferable that the plate top comprise a material that does nottransmit light so as to prevent cross-talk between wells. A highlyreflective metallic coating or constituent material may provide anespecially reflective interior surface for each of the wells so as toincrease the efficiency with which light can be transmitted tophotodetectors. An opaque white plastic material such as a plasticfilled with light scattering particles (e.g., lead oxide, alumina,silica or, preferably, titanium dioxide particles) may provide aninterior surface for the wells that is highly light scattering therebyimproving light gathering efficiency. In one embodiment, preferred platetops comprise plastics (e.g., well walls) comprising such lightscattering particles at a concentration of from 4-20 wt %, preferably6-20%, more preferably 6-15%, even more preferably 6-12%, and mostpreferred approximately 9% or 10%. In an alternate preferred embodiment,the plate top comprises an opaque, preferably non-reflective, blackmaterial to prevent the reflection or scattering of ECL-generated lightfrom different locations within a well and to prevent reflectiveinterference during ECL test measurements. In general, when imaginglight emitted from a well (e.g., when using a camera to produce an imageof light emitted from the well) it is advantageous that the interiorsurface of the well (e.g., as defined by a plate top) comprise anabsorptive (e.g., black) preferably non-scattering material since thedetection of scattered light will reduce the fidelity of the image. Ingeneral, when detecting light in a non-imaging mode (e.g., when a singlelight detector is used to detect all the light emitted from a well) itis advantageous that the interior surface of the well comprise areflective or highly scattering material so as to prevent the loss oflight due to adsorption of light at the well walls and to maximize thecollection of light at the detector.

Yet another aspect of the invention relates to improved materials foruse in the assay modules (e.g., plate tops, cassette parts, etc.) of theinvention, particularly assay modules used in luminescence assays. Morespecifically, the inventors have discovered improved materials for usein forming assay module components such as plate tops, which result inless background luminescence.

As described above, TiO₂ can be added to plate tops to provide a highlylight scattering surface that increases the efficiency of lightcollection from the wells and prevents cross-talk between wells. Onedrawback of the use of TiO₂ plates is a relatively long-livedluminescence (on the order of minutes) when the plate is exposed to UVor fluorescent light prior to insertion into the instrument. This lightintensity decays exponentially and thus, produces an undesirable timedependent background intensity signal. It is believed that the titaniumdioxide is the source of this light. One explanation (althoughspeculative) for the cause of this light emission is that uponexcitation with band gap light, a photogenerated electron hole isproduced in the TiO₂. The electron hole reacts with water to produce ahydroxyl radical. When the hydroxyl radical reacts with the conductionband electron of the TiO₂, light is generated.

To overcome this problem, a “wait before the read” time is preferablyused during the plate read cycle and/or optical filters are used toreduce the effect. Upon further investigation, however, applicantsdiscovered that the proper choice of TiO₂ (more specifically TiO₂ madeby certain methods) greatly reduced the background luminescence andeliminated the need for optical filters in the instrument. Morespecifically, the use of TiO₂ having a luminescence reducing coatingallows the wait time prior to measurement to be reduced to less than 2minutes, preferably less than 1 minute, more preferably less than 50seconds, even more preferably less than 40 seconds, even more preferablyless than 30 seconds, and most preferably less than about secondswithout the use of optical filters. It should be noted that the waittime depends on the algorithm for data processing as well as the signallevels characteristic of a given assay. More sophisticated algorithmsmay be employed to further reduce the waiting time caused by thedecaying luminescence of the TiO₂ (e.g., second order or exponentialfitting of the background signals).

Titanium dioxide exists in three different crystal forms: Rutile (mostcommon); Anatase (available; but less common); and Brookite (rare). Inaddition, most commercially available TiO₂ undergoes surface treatmentduring manufacture. There are two types of surface treatment: organicand inorganic. The TiO₂ may undergo either or both processes duringmanufacturing. The organic treatment is used to lower the surface energyof the TiO₂ particle so it will disperse well in polymers. Without anorganic treatment, the hydrophilic TiO₂ will not disperse, but remainsaggregated. Common organic surface treatments for TiO₂ are treatmentswith polyol (low molecular weight polyethylene glycol), silicone andpolydimethyl siloxane. The inorganic surface treatment providesdurability to the white pigment by preventing free radical breakdown byUV light. Common inorganic treatments include phosphate, alumina,zirconia and silica. Alumina and zirconia are the preferred organictreatments for protection from free radical damage.

Applicants have discovered that the use of inorganic surface treatedTiO₂ as an additive to an assay module component such as a plate topresults in reduced background luminescence. Thus, one embodiment of theinvention relates to an assay module component comprising TiO₂ which hadbeen subjected to inorganic treatment. Preferably, the inorganictreatment is selected from phosphate, alumina, zirconia and silica; evenmore preferably alumina and/or zirconia; and most preferably alumina.Thus, according to one preferred embodiment, the TiO₂ comprises aninorganic coating, preferably an alumina coating. Unintentionalluminescence may also be reduced by using filters, shorter waveforms,and/or more sophisticated data processing algorithms. For example,optical filters may be chosen that transmit the wavelength of thedesired ECL signal (preferably, from 500-800 nm, more preferably, from550-650 nm) and absorb luminescence from the plate top (preferably,light having a wavelength less than 500 nm). Alternatively, ECL isinduced using voltage waveforms that produce short but intense bursts ofECL (e.g., ramp waveforms having slopes of >1 V/s) so as to minimize theintegrated background luminescence during the ECL measurement.Alternatively, a data processing algorithm is used to subtractbackground luminescence. For example, the background luminescence ismeasured prior to an ECL measurement. ECL is measured and the backgroundluminescence is subtracted. If the decay characteristics of thebackground luminescence is known or measured, the value of backgroundluminescence used in the correction can be adjusted for the time betweenthe measurement of background luminescence and the measurement of ECL(e.g., by modeling the background luminescence as an exponential decaywith a time constant or by using a linear approximation of anexponential decay).

FIG. 2E illustrates a plate top 280 according to a preferred embodimentof the present invention. Plate top 280 comprises a plate top body 281,a top surface 282, well wall 285, and well inner surface 286. Plate top280 has one or more holes 284 defined by top surface 282 and innersurface 286. Plate top 280 is preferably has lightabsorptive/reflecting/scattering properties as described above. Holes284 are, preferably, configured as described above. Plate top 280 alsohas one or more corner recesses 287 that provide identifying physicalindicia for plate top 280. In particular, corner recesses 287 facilitatethe alignment and handling of plate top 280 and assist in distinguishingplate top 280 from other plates having different configurations ofrecessed areas along their respective peripheries. Advantageously, thedimensions and structure of plate top body 281 are preferably inaccordance with, or at least compatible with, industry standards for thefootprints and shapes of similar types of assay plates. Plate top 280,preferably, also comprises indicia 283 that may be used to identify aparticular hole 284.

FIG. 2F shows another embodiment of plate top 280. Plate top 290illustrates a plate top with a plurality of holes 291. In a preferredembodiment, holes 291 in plate top 290 have the cross sectional shape ofa square. In an alternate embodiment, holes 291 have the cross sectionalshape of a circle, and decrease in diameter as they move away from thetop of the plate. In FIG. 2F, plate top 280 has three hundred eightyfour (384) holes 291, arranged in a 2 dimensional array of rows andcolumns. FIG. 2G shows another embodiment of plate top 280. Plate top295 illustrates a plate top with an array of holes 297. In a preferredembodiment, the holes 297 in plate top 295 have the cross sectionalshape of a circle. In an alternate embodiment, holes 297 have the crosssectional shape of a square. In FIG. 2G, plate top 295 has 1536 holes.

The invention also includes assay module tops and assembled assaymodules comprising an assay module top and a plate bottom or assaymodule substrate. The assay module top may be a plate top (as describedabove). The assay module top may have, e.g., holes, channels, and/orwells that when mated to a plate bottom or assay module substrate definewells and/or chambers, such wells and/or chambers preferably comprisingone or more electrodes (and/or assay domains) provided by the platebottom or assay module substrate. The assay module top may haveadditional channels, tubes or other microfluidics so as to allow theflow of samples into, out of and/or between wells, flow cells andchambers of an assay module.

5.1.5 Electrode/Contact Configurations

Another aspect of the invention relates to novel electrode and/orcontact configurations. According to the invention, the shape,composition, placement/location, configuration, pattern, thickness,surface properties and many other characteristics of the electrodes andcontacts are optimized to result in improved methods and systems.

Optimizing the configuration of electrodes allows for (i) higher densityassay arrays, (ii) the reduction of the variation in voltage across aplurality of wells and/or assay domains; (iii) the division of an assaymodule into independently addressable portions (e.g., allowing forindependently addressable sectors of jointly addressable wells on amulti-well assay plate); and/or (iv) ease of manufacture.

Optimizing the configuration of contacts (e.g., electrical contacts onthe bottom of an assay module substrate and/or assay plate bottom)allows for: (i) reducing the number of necessary electrical connectors;(ii) reducing the variation in voltage across a plurality of wellsand/or assay domains; (iii) controlling any flexing or bending of thewell bottom during contacting; (iv) the division of an assay module intoindependently addressable portions (e.g., allowing for independentlyaddressable sectors of jointly addressable wells on a multi-well assayplate); and/or (iv) ease of manufacture.

One embodiment of the invention relates to a multi-well plate comprisinga plate top having a plurality of rows of openings and a plate bottomhaving first electrode strips (preferably working electrode strips) andsecond electrode strips (preferably counter electrode strips) patternedthereon, wherein the plate top is affixed on the substrate therebyforming a plurality of rows of wells from the openings, wherein thebottom of each well comprises an exposed portion of at least one firstelectrode strip and two exposed edge portions of the second electrodestrips. More specifically, referring to FIG. 10A, working electrodestrips 1052 and counter electrode strips 1054 are arranged on a platebottom so that when the plate bottom is adjoined to the plate top, theworking electrode strip is centered within each well, with a portion oftwo adjacent counter electrodes on each side.

Another embodiment of the invention relates to a multi-well platecomprising a plate top having a plurality of rows of openings and asubstrate, wherein the plate top is placed on the substrate therebyforming a plurality of well rows from the plurality of openings and wellbottoms, the well bottoms comprising a center portion of a workingelectrode strip and a portion from two counter electrode strips on eachside of the portion of the working electrode strip. Preferably, the wellrows are aligned with the working electrode strips and the counterelectrode strips, wherein each of the plurality of well rows comprises:(i) a first well comprising a first well bottom including an exposedportion of a first working electrode strip (preferably centered withinthe well), a first edge portion including an exposed portion of a firstcounter electrode strip and a second edge portion including an exposedportion of a second counter electrode strip and (ii) at least a secondwell comprising a second well bottom including an exposed portion of thefirst working electrode strip (preferably centered), a first edgeportion including an exposed portion of the first counter electrodestrip and a second edge portion including an exposed portion of thesecond counter electrode strip. See, FIGS. 10A and 16A.

Another aspect of the invention relates to an assay module preferablyhaving wells and/or chambers, most preferably a multi-well plate,comprising a substrate having a first side and a second side, thesubstrate comprising a plurality of first electrode surfaces (preferablyworking electrode surfaces) and, preferably, a plurality of secondelectrode surfaces (preferably counter electrode surfaces) on the firstside and one or more conductive contacts on the second side, wherein twoor more, preferably all or substantially all, of the plurality of wellsand/or chambers each comprise one or more working electrode surfaces andone or more counter electrode surfaces.

One embodiment of the invention relates to a multi-well plate, whereinthe plate substrate includes one or more conductive contacts adapted to:(a) distribute voltage, applied to the conductive contacts, uniformlythroughout the plurality of wells, preferably distribute voltage suchthat any voltage variation is less than 0.5 volts, more preferably lessthan 0.1 volts, even more preferably less than 0.01 volts; (b)distribute voltage uniformly throughout the plurality of wells such thatthe variation of the sum of the effective resistance from the contactsto the counter electrode and the effective resistance from the contactsto working electrode for the plurality of wells is less than 10 ohms,preferably less than 5 ohms, more preferably less than 1 ohms, and mostpreferred constant; and/or (c) distribute voltage uniformly throughoutthe plurality of wells such that the variation of V_(c) minus V_(w) forthe plurality of wells is less than 0.5 volts, preferably the variationof Vc−Vw is less than 0.1 volt, most preferably less than 50 mvolts,where Vc and Vw are defined as the voltage at the counter electrode andthe voltage at the working electrode, respectively.

Preferably, the plates comprise plate contacts adapted to uniformlydistribute current and/or voltage to the wells. According to oneembodiment, the plate substrate further comprises a bottom surfacecomprising at least one independent electrical contact surface that iselectrically connected to each of the plurality of independentlyaddressable sectors of jointly addressable wells. The plate or platesubstrate may further comprise one or more common (to more than onesector) electrical contact surfaces located on a surface of the plate,preferably, on the bottom of the plate substrate (e.g., there may be oneor more common electrical contact surfaces that are connected to acounter electrode surface that is common to the entire plate).Advantageously, the electrical contact locations are positioned on thebottom surface between the plurality of wells such that the apparatuscontacts contact the plate between the wells. According to oneembodiment, the bottom surface comprises between 2 and 10 electricalcontact surfaces per sector, even more preferably the bottom surfacecomprises two, six or seven contact surfaces per sector.

According to one embodiment, the plate bottom or substrate comprises abottom surface comprising a plurality of electrical contacts or contactlocations, preferably an array of electrical contact locations arrangedin a 2×3 array.

The term “contact locations” is used herein to refer to the actuallocations of the assay plate where the electrical connectors from theapparatus contact the plate. The term “contacts” or “contact surfaces”is intended to refer to the conductive surfaces on the plate bottomwhich are contacted with the electrical connections or electricalconnectors. The “contact locations” are located within the “contactsurfaces”. The area of the “contact surface” can be significantly largerthan that of the “contact locations”. For example, referring to FIG.10A, a plate bottom is illustrated showing working contacts 1072 (whichare electrically connected to working electrodes 1052 via conductivethrough-holes 1062) and counter contacts 1074 (which are electricallyconnected to counter electrodes 1054 via conductive through-boles 1064).This figure shows an embodiment where the elongated counter contactsurface 1074 is larger, yet encompasses the locations which arecontacted by the counter electrical connector of the apparatus (e.g.,the preferred counter electrode contact locations 3470 as shown in FIG.34B).

Preferably, each of the electrical contact locations is positionedbetween 0.1 and 1 inches away from each adjacent electrical contactlocation, more preferably between 0.2 to 0.8 inches, even morepreferably 0.3 to 0.4 inches.

Preferably, contact surfaces on the bottom of the plate comprise contactlocations that are located between wells of the plate so that contactingthe contact locations with an electrical connector does not distort thebottom surface of a well. FIG. 34A, shows (with respect to a fullyassembled multi-well plate 3400 shown having wells 3405 arranged in astandard 96 well plate configuration) preferred contact locations on theplate bottom of plate 3400. Plate 3400 has an array, preferably a 2×3array, of square sectors or regions 3410 (the division into sectorsrepresented by dotted lines), wherein each sector comprises one or moreelectrical contact locations 3420 (represented by X's) and 3430(represented by *'s) on a bottom surface of the plate bottom, thecontact locations being located between wells on plate 3400. The contactlocations on each sector are, preferably, arranged in a 2×3 array.Electrical contact locations 3420 are, preferably, connected to workingelectrodes; electrical contact locations 3430 are, preferably, connectedto counter electrodes. The electrical contacts locations are located atat least one, preferably at least two, more preferably at least four andmost preferably all, of the following locations, the locations beingdefined by coordinates (X, Y) measured (inches, ±0.250″, preferably+0.125″) from the left and top edges, respectively, of the plate(viewing the plate from above, i.e., referring to FIG. 1, well A1 beingthe closest to the top left corner).

-   -   (i) one or more (preferably two or more, more preferably three        or more and most preferably all) of first sector locations:        (0.743, 0.620), (1.097, 0.620), (1.451, 0.620), (0.743, 1.329),        (1.097, 1.329), (1.451, 1.329), most preferably, one or more        working electrode contact locations selected from (0.743,        0.620), (1.451, 0.620), (0.743, 1.329), and (1.451, 1.329)        and/or one or more counter electrode contact locations selected        from (1.097, 0.620), and (1.097, 1.329);    -   (ii) one or more (preferably two or more, more preferably three        or more and most preferably all) of second sector locations:        (2.161, 0.620), (2.515, 0.620), (2.869, 0.620), (2.161, 1.329),        (2.515, 1.329), (2.869, 1.329), most preferably, one or more        working electrode contact locations selected from (2.161,        0.620), (2.869, 0.620), (2.161, 1.329), and (2.869, 1.329)        and/or one or more counter electrode contact locations selected        from (2.515, 0.620), and (2.515, 1.329);    -   (iii) one or more (preferably two or more, more preferably three        or more and most preferably all) of third sector locations:        (3.579, 0.620), (3.933, 0.620), (4.287, 0.620), (3.579, 1.329),        (3.933, 1.329), (4.287, 1.329), most preferably, one or more        working electrode contact locations selected from (3.579,        0.620), (4.287, 0.620), (3.579, 1.329), and (4.287, 1.329)        and/or one or more counter electrode contact locations selected        from (3.933, 0.620), and (3.933, 1.329);    -   (iv) one or more (preferably two or more, more preferably three        or more and most preferably all) of fourth sector locations:        (0.743, 2.038), (1.097, 2.038), (1.451, 2.038), (0.743, 2.747),        (1.097, 2.747), (1.451, 2.747), most preferably, one or more        working electrode contact locations selected from (0.743,        2.038), (1.451, 2.038), (0.743, 2.747), and (1.451, 2.747)        and/or one or more counter electrode contact locations selected        from (1.097, 2.038), and (1.097, 2.747);    -   (v) one or more (preferably two or more, more preferably three        or more and most preferably all) of fifth sector locations:        (2.161, 2.038), (2.515, 2.038), (2.869, 2.038), (2.161, 2.747),        (2.515, 2.747), (2.869, 2.747), most preferably, one or more        working electrode contact locations selected from (2.161,        2.038), (2.869, 2.038), (2.161, 2.747), and (2.869, 2.747)        and/or one or more counter electrode contact locations selected        from (2.515, 2.038), and (2.515, 2.747); and    -   (vi) one or more (preferably two or more, more preferably three        or more and most preferably all) of sixth sector locations:        (3.579, 2.038), (3.933, 2.038), (4.287, 2.038), (3.579, 2.747),        (3.933, 2.747), (4.287, 2.747), most preferably, one or more        working electrode contact locations selected from (3.579,        2.038), (4.287, 2.038), (3.579, 2.747), and (4.287, 2.747)        and/or one or more counter electrode contact locations selected        from (3.933, 2.038), and (3.933, 2.747).

The pattern of contact locations described above is illustrated in FIG.34A in relation to a 96-well plate, however, it is not limited to usewith 96-well plates and may be applied to plates or plate bottoms ofmany plate formats including 1, 2, 6, 24, 384, 1536, 6144 and 9600-wellplates. Preferably, the contact locations are located in the regionsbetween the wells of a fully assembled plate.

The preferred locations of contact locations may also be specified inrelation to the location of wells in a fully assembled plate. Apreferred embodiment relates to a 96 well plate having electrodes andelectrical contact surfaces. Referring to FIG. 1, the 96 well platecomprises rows (designated with the letters A through H) and columns ofwells (designated with the numbers 1-12). The plate preferably comprisesone or more, preferably two or more, more preferably all, of thefollowing sectors (as shown in FIG. 34A):

-   -   a first sector comprising wells A1 through A4, B1 through B4, C1        through C4, and D1 though D4;    -   a second sector comprising wells A5 through A8, B5 through B8,        C5 through C8, and D5 though D8;    -   a third sector comprising wells A9 through A12, B9 through B12,        C9 through C12, and D9 through D12;    -   a fourth sector comprising wells E1 through E4, F1 through F4,        G1 through G4, and H1 though H4;    -   a fifth sector comprising wells E5 through E8, F5 through F8, G5        through G8, and H5 though H8; and    -   a sixth sector comprising wells E9 through B12, F9 through F1,        G9 through G12, and H9 though H12.

Each of the designations refers to a region of the plate defined by therow and column. For example, A1 refers to the well in row A andcolumn 1. We use the following notation herein to refer to the regionbetween wells: “well1-well2”. For example, the term “A1-B2” is usedherein to refer to the region between well A1 (row A, column 1) and B2(row B, column 2). The term “A1 through A4” is used to refer to theregion including wells A1, A2, A3 and A4, including the spacein-between.

According to one preferred embodiment of the invention, the sectorcomprises one or more electrical contact locations or contact surfaceson the plate bottom at one or more, preferably two or more, morepreferably four or more and most preferred six of the following sectorlocations (by reference to FIGS. 1 and 34A):

-   -   (i) one or more, more preferably two or more and most preferred        six, of first sector locations: A1-B2; A2-B3; A3-B4; C1-D2;        C2-D3; C3-D4, most preferably, one or more working electrode        contact locations selected from A1-B2, A3-B4, C1-D2 and C3-D4        and/or one or more counter electrode contact locations selected        from A2-B3 and C2-D3;    -   (ii) one or more, more preferably two or more and most preferred        six, of second sector locations: A5-B6; A6-B7; A7-B8; C5-D6;        C6-D7; C7-D8, most preferably, one or more working electrode        contact locations selected from A5-B6, A7-B8, C5-D6 and C7-D8        and/or one or more counter electrode contact locations selected        from A6-B7 and C6-D7;    -   (iii) one or more, more preferably two or more and most        preferred six, of third sector locations: A9-B10; A10-B11;        A11-B12; C9-D10; C10-D11; C11-D12, most preferably, one or more        working electrode contact locations selected from A9-B10,        A11-B12, C9-D10 and C11-D12 and/or one or more counter electrode        contact locations selected from A10-B11 and C10-D11;    -   (iv) one or more, more preferably two or more and most preferred        six, of fourth sector locations: E1-F2; E2-F3; E3-F4; G1-H2;        G2-H3; G3-H4, most preferably, one or more working electrode        contact locations selected from E1-F2, E3-F4, G1-H2 and G3-H4        and/or one or more counter electrode contact locations selected        from E2-F3 and G2-H3;    -   (v) one or more, more preferably two or more and most preferred        six, of fifth sector locations: E5-F6; E6-F7; E7-F8; G5-H6;        G6-H7; G7-H8, most preferably, one or more working electrode        contact locations selected from E5-F6, E7-F8, G5-H6 and G7-H8        and/or one or more counter electrode contact locations selected        from E6-F7 and G6-H7; and    -   (vi) one or more, more preferably two or more and most preferred        six, of sixth sector locations: E9-F10; B10-F11; E11-F12;        G9-H10; G10-H11; G11-H12, most preferably, one or more working        electrode contact locations selected from E9-F10; E11-F12;        G9-H10 and G11-H12 and/or one or more counter electrode contact        locations selected from E10-F11 and G10-H11.

By analogy, preferred contact location(s) on a 384-well plate or platebottom for use with a 384 well plate may be defined in relationship tothe wells of a 384-well plate having a standard configuration of wellsin rows A-P and columns 1-24. In one embodiment, the plate, preferably,comprises one or more, preferably two or more, more preferably all, ofthe following sectors:

-   -   a first sector comprising wells A1 through A5, B1 through B8, C1        through C8, D1 though D8, E1 through E8, F1 through F8, G1        through G8, and H1 though H8;    -   a second sector comprising wells A9 through A16, B9 through B16,        C9 through C16, D9 though D16, E9 through E16, F9 through F16,        G9 through G16, and H9 though H16;    -   a third sector comprising wells A17 through A24, B17 through        B24, C17 through C24, D17 though D24, E17 through E24, F17        through F24, G17 through G24, and H17 though H24;    -   a fourth sector comprising wells I17 through I18, J1 through J8,        K1 through K8, L1 though L8, M1 through M8, N1 through N8, O1        through O8 and P1 through P8;    -   a fifth sector comprising wells I9 through I16, J9 through J16,        K9 through K16, L9 though L16, M9 through M16, N9 through N16,        O9 through O16 and P9 through P16; and    -   a sixth sector comprising wells I17 through I24, J17 through        J24, K17 through K24, L17 though L24, M17 through M24, N17        through N24, O17 through O24 and P17 through P24.

Preferably, each plate sector comprises one or more electrical contactlocations at one or more, preferably two or more, more preferably fouror more and most preferred six, of the following locations:

-   -   (i) one or more, preferably two or more, more preferably four or        more and most preferred all, of first sector locations: B2-C3;        B4-C5; B6-C7; F2-G3; F4-G5; F6-G7, most preferably, one or more        working electrode contact locations selected from B2-C3, B6-C7,        F2-G3 and F6-G7 and/or one or more counter electrode contact        locations selected from B4-C5 and F4-G5;    -   (ii) one or more, preferably two or more, more preferably four        or more and most preferred all, of second sector locations:        B10-C11; B12-C13; B14-C15; F10-G11; F12-G13; F14-G15, most        preferably, one or more working electrode contact locations        selected from B10-C11, B14-C15, F10-G111 and F14-G15 and/or one        or more counter electrode contact locations selected from        B12-C13 and F10-G11;    -   (iii) one or more, preferably two or more, more preferably four        or more and most preferred all, of third sector locations:        B18-C19; B20-C21; B22-C23; F18-G19; F20-G21; F22-G23, most        preferably, one or more working electrode contact locations        selected from B18-C19, B22-C23, F18-G19 and F22-G23 and/or one        or more counter electrode contact locations selected from        B20-C21 and F20-G21;    -   (iv) one or more, preferably two or more, more preferably four        or more and most preferred all, of fourth sector locations:        J2-K3; J4-K5; J6-K7; N2-O3; N4-O5; N6-O7, most preferably, one        or more working electrode contact locations selected from J2-K3,        J6-K7, N2-O3 and N6-O7 and/or one or more counter electrode        contact locations selected from J4-K5 and N4-O5;    -   (v) one or more, preferably two or more, more preferably four or        more and most preferred all, of fifth sector locations: J10-K11;        J12-K13; J14-K15; N10-O11; N12-O13; N14-O15, most preferably,        one or more working electrode contact locations selected from        J10-K11, J14-K5, N10-O11 and N14-O15 and/or one or more counter        electrode contact locations selected from J12-K13 and J14-K15;        and    -   (vi) one or more, preferably two or more, more preferably four        or more and most preferred all, of sixth sector locations:        J18-K19; J20-K21; J22-K23; N18-O19; N20-O21; N22-O23, most        preferably, one or more working electrode contact locations        selected from J18-K19, J22-K23, N18-O19 and N22-O23 and/or one        or more counter electrode contact locations selected from        J20-K21 and N20-O21.

FIG. 34B, another embodiment of the invention, shows (with respect to afully assembled multi-well plate 3450 shown having wells 3455 arrangedin a standard 96 well plate configuration) preferred contact locationson the plate bottom of plate 3450, the plate having a differentarrangement of sectors than plate 3400. Plate 3450 has an array,preferably a 1×12 array, of columnar sectors or regions 3460 (thedivision into sectors represented by dotted lines), wherein each sectorcomprises one or more electrical contact locations 3480 (represented byX's) and 3470 (represented by *'s) on a bottom surface of the platebottom, the contact locations being located between wells on plate 3450.The contact locations on each sector are, preferably, arranged in a 7×1array. Electrical contact locations 3480 are, preferably connected toworking electrodes; electrical contact locations 3470 are, preferably,connected to counter electrodes. The electrical contacts are located atat least one, preferably at least two, more preferably at least four andmost preferably all, of the following locations, the locations beingdefined by coordinates (X, Y) measured (inches, +0.250″, preferably+0.125″) from the left and top edges, respectively, of the plate(viewing the plate from above, i.e., referring to FIG. 1, well A1 beingthe closest to the top left corner):

-   -   (i) one or more (preferably two or more, more preferably three        or more and most preferably all) of first sector locations:        (0.566, 0.620), (0.566, 0.975), (0.566, 1.329), (0.566, 1.684),        (0.566, 2.038), (0.566, 2.393), (0.566, 2.747), most preferably,        one or more working electrode contact locations selected from        (0.566, 0.620), (0.566, 1.329), (0.566, 2.038) and (0.566,        2.747) and/or one or more counter electrode contact locations        selected from (0.566, 0.975), (0.566, 1.684) and (0.566, 2.393);    -   (ii) one or more (preferably two or more, more preferably three        or more and most preferably all) of second sector locations:        (0.920, 0.620), (0.920, 0.975), (0.920, 1.329), (0.920, 1.684),        (0.920, 2.038), (0.920, 2.393), (0.920, 2.747), most preferably,        one or more working electrode contact locations selected from        (0.920, 0.620), (0.920, 1.329), (0.920, 2.038) and (0.920,        2.747) and/or one or more counter electrode contact locations        selected from (0.920, 0.975), (0.920, 1.684) and (0.920, 2.393);    -   (iii) one or more (preferably two or more, more preferably three        or more and most preferably all) of third sector locations:        (1.275, 0.620), (1.275, 0.975), (1.275, 1.329), (1.275, 1.684),        (1.275, 2.038), (1.275, 2.393), (1.275, 2.747), most preferably,        one or more working electrode contact locations selected from        (1.275, 0.620), (1.275, 1.329), (1.275, 2.038) and (1.275,        2.747) and/or one or more counter electrode contact locations        selected from (1.275, 0.975), (1.275, 1.684) and (1.275, 2.393);    -   (iv) one or more (preferably two or more, more preferably three        or more and most preferably all) of fourth sector locations:        (1.629, 0.620), (1.629, 0.975), (1.629, 1.329), (1.629, 1.684),        (1.629, 2.038), (1.629, 2.393), (1.629, 2.747), most preferably,        one or more working electrode contact locations selected from        (1.629, 0.620), (1.629, 1.329), (1.629, 2.038) and (1.629,        2.747) and/or one or more counter electrode contact locations        selected from (1.629, 0.975), (1.629, 1.684) and (1.629, 2.393);    -   (v) one or more (preferably two or more, more preferably three        or more and most preferably all) of fifth sector locations:        (1.983, 0.620), (1.983, 0.975), (1.983, 1.329), (1.983, 1.684),        (1.983, 2.038), (1.983, 2.393), (1.983, 2.747), most preferably,        one or more working electrode contact locations selected from        (1.983, 0.620), (1.983, 1.329), (1.983, 2.038) and (1.983,        2.747) and/or one or more counter electrode contact locations        selected from (1.983, 0.975), (1.983, 1.684) and (1.983, 2.393);    -   (vi) one or more (preferably two or more, more preferably three        or more and most preferably all) of sixth sector locations:        (2.338, 0.620), (2.338, 0.975), (2.338, 1.329), (2.338, 1.684),        (2.338, 2.038), (2.338, 2.393), (2.338, 2.747), most preferably,        one or more working electrode contact locations selected from        (2.338, 0.620), (2.338, 1.329), (2.338, 2.038) and (2.338,        2.747) and/or one or more counter electrode contact locations        selected from (2.338, 0.975), (2.338, 1.684) and (2.338, 2.393);    -   (vii) one or more (preferably two or more, more preferably three        or more and most preferably all) of seventh sector locations:        (2.692, 0.620), (2.692, 0.975), (2.692, 1.329), (2.692, 1.684),        (2.692, 2.038), (2.692, 2.393), (2.692, 2.747), most preferably,        one or more working electrode contact locations selected from        (2.692, 0.620), (2.692, 1.329), (2.692, 2.038) and (2.692,        2.747) and/or one or more counter electrode contact locations        selected from (2.692, 0.975), (2.692, 1.684) and (2.692, 2.393);    -   (viii) one or more (preferably two or more, more preferably        three or more and most preferably all) of eighth sector        locations: (3.046, 0.620), (3.046, 0.975), (3.046, 1.329),        (3.046, 1.684), (3.046, 2.038), (3.046, 2.393), (3.046, 2.747),        most preferably, one or more working electrode contact locations        selected from (3.046, 0.620), (3.046, 1.329), (3.046, 2.038) and        (3.046, 2.747) and/or one or more counter electrode contact        locations selected from (3.046, 0.975), (3.046, 1.684) and        (3.046, 2.393);    -   (ix) one or more (preferably two or more, more preferably three        or more and most preferably all) of ninth sector locations:        (3.400, 0.620), (3.400, 0.975), (3.400, 1329), (3.400, 1.684),        (3.400, 2.038), (3.400, 2.393), (3.400, 2.747), most preferably,        one or more working electrode contact locations selected from        (3.400, 0.620), (3.400, 1.329), (3.400, 2.038) and (3.400,        2.747) and/or one or more counter electrode contact locations        selected from (3.400, 0.975), (3.400, 1.684) and (3.400, 2.393);    -   (x) one or more (preferably two or more, more preferably three        or more and most preferably all) of tenth sector locations:        (3.755, 0.620), (3.755, 0.975), (3.755, 1.329), (3.755, 1.684),        (3.755, 2.038), (3.755, 2.393), (3.755, 2.747), most preferably,        one or more working electrode contact locations selected from        (3.755, 0.620), (3.755, 1.329), (3.755, 2.038) and (3.755,        2.747) and/or one or more counter electrode contact locations        selected from (3.755, 0.975), (3.755, 1.684) and (3.755, 2.393);    -   (xi) one or more (preferably two or more, more preferably three        or more and most preferably all) of eleventh sector locations:        (4.109, 0.620), (4.109, 0.975), (4.109, 1.329), (4.109, 1.684),        (4.109, 2.038), (4.109, 2.393), (4.109, 2.747), most preferably,        one or more working electrode contact locations selected from        (4.109, 0.620), (4.109, 1.329), (4.109, 2.038) and (4.109,        2.747) and/or one or more counter electrode contact locations        selected from (4.109, 0.975), (4.109, 1.684) and (4.109, 2.393);        and    -   (xii) one or more (preferably two or more, more preferably three        or more and most preferably all) of twelfth sector locations:        (4.463, 0.620), (4.463, 0.975), (4.463, 1.329), (4.463, 1.684),        (4.463, 2.038), (4.463, 2.393), (4.463, 2.747), most preferably,        one or more working electrode contact locations selected from        (4.463, 0.620), (4.463, 1.329), (4.463, 2.038) and (4.463,        2.747) and/or one or more counter electrode contact locations        selected from (4.463, 0.975), (4.463, 1.684) and (4.463, 2.393).

The contact locations of a 96-well plate or plate bottom having a 1×12array of sectors may also be defined in relationship to the position ofthe wells in the fully assembled plate. Preferably, at least one and,most preferably, all of the sectors have one or more (preferably two ormore, more preferably three or more and most preferably all) contactlocations selected from: An-Bn, Bn-Cn, Cn-Dn, Dn-En, En-Fn, Fn-Gn andGn-Hn where n is the number designating the plate column defining thesector (by reference to FIG. 1), and most preferably has at least oneworking contact location selected from An-Bn, Cn-Dn, En-Fn, and Gn-Hnand at least one counter contact location selected from Bn-Cn, Dn-En andFn-Gn.

According to preferred embodiments of the invention, theabove-identified plate bottoms having the 2×3 or 1×12 array of sectorsand contact locations defined on said sectors further comprise a platetop having a plurality of openings forming a plurality of wells alignedwith the electrodes.

The invention also relates to an apparatus configured to measureluminescence from a multi-well plate having the above-identified contactconfiguration. More specifically, comprising electrical connectors tocontact the plate bottom at the above-identified contact locations. Theinvention also relates to methods of performing assays comprising thestep of contacting the assay plate at the above-identified contactlocations.

According to one embodiment, the apparatus comprises a plurality ofelectrical connectors, wherein the plurality of electrical connectors isconfigured to contact the bottom surface, preferably between the wells.Preferably, the plurality of electrical connectors comprises one or moreworking connectors (i.e., the electrical connector which contacts theplate to electrically connect the source of electrical energy to theworking electrodes) and one or more counter connectors (i.e., theelectrical connectors which contact the plate to electrically connectthe source of electrical energy to the counter electrodes).

Preferably, each sector is contacted by the plurality of electricalconnectors at six locations, more preferably a 2×3 array of locations asdefined above.

Another preferred embodiment of the invention relates to an apparatuscomprising a light detector adapted to measure luminescence emitted fromthe plurality of wells and a plurality of electrical connectors, whereinthe plurality of electrical connectors are configured to contact thebottom surface of a multi-well plate, preferably of a 384 well plate atthe above-described contact locations.

Another aspect of the invention relates to plate bottom (andcorresponding multi-well plates) having a plurality of electrodes on afirst surface and a plurality of “contact sectors” on a second surface.The term “contact sector” is used herein to refer to independentlyaddressable regions or sectors of contacts. FIG. 11A illustrates anexample of a contact sector 1170 comprising a working contact surface1172 and a counter electrode surface 1174.

Accordingly, another embodiment of the invention relates to a multi-wellplate bottom and/or multi-well plate comprising:

-   -   (a) a substrate having a top surface and a bottom surface;    -   (b) a plurality of patterned working electrodes on the top        surface;    -   (c) a plurality of patterned counter electrodes on the top        surface, each of the patterned counter electrodes being        associated with corresponding patterned working electrodes; and    -   (d) two or more independently addressable contact sectors on the        bottom surface, each of the contact sectors corresponding to an        electrode sector comprising one or more of the plurality of        patterned working electrodes and one or more of the plurality of        patterned counter electrodes on the top surface and including a        plurality of conductive contact surfaces.

Preferably, the plurality of conductive contact surfaces include:

-   -   (i) a first conductive contact surface located within a first        contact region, the first conductive contact surface being        electrically connected to the one or more corresponding        patterned working electrodes on the top surface; and    -   (ii) a second conductive contact surface located within a second        contact region, the second conductive contact surface being        electrically connected to the one or more corresponding        patterned counter electrodes on the top surface;    -   wherein the first conductive contact surface and the second        conductive contact surface are electrically isolated from each        other.

Preferably, the two or more sectors comprise at least six sectors, morepreferably six sectors in a 2×3 array of equal size sectors.

According to a preferred embodiment, the substrate further comprises:(i) first conductive through-holes electrically connecting the firstconductive contact surface on the bottom surface with the one or morecorresponding patterned working electrodes on the top surface and (ii)second conductive through-holes electrically connecting the secondconductive contact surface to the one or more corresponding patternedcounter electrodes on the top surface.

Preferably, the first contact region has a U-shaped configuration andthe second contact region has a T-shaped configuration, wherein theU-shaped configuration is mated with the T-shaped configuration withinthe sector. See FIGS. 11A and 12A. Table I (below) gives, preferredlocations of said U-shaped first contact region and said T-shaped secondcontact region as defined with relation to the positions of wells in afully assembled 96-well plate (having the standard configuration of rowsA through H and columns 1 through 12) and a fully assembled 384-wellplate (having the standard configuration of rows A through P and columns1 through 24). The table describes the contact regions by dividing theminto segments that are roughly aligned with lines, the endpoints of thelines being defined in relation ship to wells on the plates.

Referring to FIGS. 11A and 12A, preferred embodiments include 96-wellplates or 384-well plates having a standard configuration of wells (orplate bottoms for said 96-well or 384-well plates), the multi-wellplates (or plate bottoms) comprising:

a substrate having a top surface and a bottom surface;

a plurality of patterned working electrodes on the top surface;

a plurality of patterned counter electrodes on the top surface, each ofthe patterned counter electrodes being associated with correspondingpatterned working electrodes; and

one or more, preferably six, independently addressable contact sectorson the bottom surface (e.g., one or more, preferably all, of the contactsectors as listed in Table I), each of the contact sectors correspondingto an electrode sector comprising one or more of the plurality ofpatterned working electrodes on the top surface and one or more of theplurality of patterned counter electrodes on the top surface, the one ormore independently addressable contact sectors including a plurality ofconductive contact surfaces;

-   -   preferably, wherein the plurality of conductive contact surfaces        for a given contact sector include:        -   (i) a first conductive contact surface located within a            first contact region, the first contact region having a            U-shaped configuration and comprising segments aligned as            defined in Table I, wherein the first conductive contact            surface is electrically connected (preferably, via one or,            more preferably, a plurality of conductive through-holes            through said substrate, the through-holes, preferably, being            located within the area defined by said first contact            region) to the one or more corresponding patterned working            electrodes on the top surface; and        -   (ii) a second conductive contact surface located within a            second contact region, the second contact region having a            T-shaped configuration and comprising segments as defined in            Table I, wherein the second conductive contact surface is            electrically connected (preferably, via one or, more            preferably, a plurality of conductive through-holes through            said substrate, the through-holes, preferably, being located            within the area defined by said second contact region) to            the one or more corresponding patterned counter electrodes            on the top surface;            -   wherein the first conductive contact surface and the                second conductive contact surface are electrically                isolated from each other.

Other embodiments of the invention would include rotating any one of theabove-identified U and/or T configurations in any one or more of theabove sectors. For example, rotating either the U and/or the T 90degrees, 180 degrees or the like.

TABLE I Table of Preferred Locations of Contact Regions on Bottom ofPlate Bottom. Contact regions are described as comprising segmentsroughly aligned with lines on the plate bottom, the endpoints of thelines being defined by relationship to the position of the wells of theplate. Preferred Location of Contact Region (describing the contactregions Contact Contact as comprising segments roughly aligned with thefollowing lines) Sector Region Plate is 96-Well Plate Plate is 384-WellPlate 1 First A1-B2 to D1-D2, A3-B4 to D3-D4 B2-C3 to H2-H3, B6-C7 toH6-H7 and D1 to D4 and H1 to H8 Second Al to A4 and A2-A3 to C2-D3 A1 toA8 and A4-A5 to F4-G5 2 First A5-B6 to D5-D6, A7-B8 to D7-D8 B10-C11 toH10-H11, B14-C15 to and D5 to D8 H14-H15 and H9 to H16 Second A5 to A8and A6-A7 to C6-D7 A9 to A16 and A12-A13 to F12-G13 3 First A9-B10 toD9-D10, A11-B12 to D11- B18-C19 to H18-H19, B22-C23 D12 and D9 to D12 toH22-H23 and H17 to H24 Second A9 to A12 and A10-A11 to C10-D11 A17 toA24 and A20-A21 to F20-G21 4 First G1-H2 to E1-E2, G3-H4 to N2-O3 toI2-I3, N6-O7 to I6-I7 E3-E4 and E1 to E4 and I1 to I8 Second H1 to H4and H2-H3 to E2-F3 P1 to P8 and P4-P5 to J4-K5 5 First G5-H6 to E5-E6,G7-H8 to E7-E8 and N10-O11 to I10-I11, N14-O15 to I14- E5 to E8 I15 andI9 to I16 Second H5 to H8 and H6-H7 to E6-F7 P9 to P16 and P12-P13 toJ12-K13 6 First G9-H10 to E9-E10, G11-H12 to N18-O19 to I18-I19, N22-O23to I22- E11-E12 and E9 to E12 I23 and I17 to I24 Second H9 to H12 andH10-H11 to E10-F11 P17 to P24 and P20-P21 to J20-K21 The notation A1refers to well A1. The notation A1-B2 refers to the region midwaybetween wells A1 and B2. The notation A2-A3 to C2-D3, therefore, refersto the region to a line starting midway between wells A2 and A3 andending midway between wells C2 and D3.

Referring again to FIGS. 11A and 12A, another embodiment of theinvention relates to a multi-well plate bottom or assay substrate and/oran assay module containing said plate bottom or assay substrate(preferably, a multi-well plate bottom and/or a multi-well platecontaining said plate bottom), said plate bottom or assay substratecomprising:

-   -   (a) a substrate divided into a 2×3 array of sectors, the        substrate having a top surface and a bottom surface, each sector        having an array of assay regions defined by columns and rows;    -   (b) one or more patterned working electrodes on the top surface        within each sector, each sector comprising elongated working        electrodes being aligned with the array columns;    -   (c) one or more patterned counter electrodes on the top surface        within each sector, each sector comprising elongated counter        electrodes being aligned with the array columns and being        electrically isolated from and located between the elongated        working electrodes; and    -   (d) one or more contacts on the bottom surface of each sector.

Preferably, each assay region defines a surface of a well or chamber inthe fully assembled assay module. Most preferably, the array of assayregions corresponds to the configuration of wells in a standard 96-wellplate or a standard 384-well and the elongated counter electrodescomprise widened electrode areas adjacent and between the assay regions(the narrow regions match the resistance of the working electrode areaswhile the widened areas ensure that the surface of the counterelectrodes are exposed in wells of the plate) and/or the elongatedcounter electrodes are electrically connected with an elongatedconnector perpendicular and adjacent one end of each elongated counterelectrode within the sector. Preferably, each assay region overlaps aportion of at least two elongated counter electrodes and a portion ofone elongated working electrode.

One embodiment of the invention relates to an assay module or assaymodule element (preferably a multi-well plate or multi-well platebottom) comprising:

-   -   (a) a substrate having a top surface and a bottom surface;    -   (b) a plurality of working electrodes (preferably patterned) on        the top surface;    -   (c) a plurality of counter electrodes (preferably patterned) on        the top surface, each of the counter electrodes being associated        with corresponding working electrodes; and    -   (d) two or more independently addressable sectors, each sector        having two or more independently addressable contacts on the        bottom surface, each of the contacts corresponding to one or        more electrodes within assay regions or well regions (e.g., the        surface of the substrate which forms part of the well bottom        after attaching the plate top) within one of the sectors.

Preferably, the sectors include at least six, preferably at least twelvelinear sectors. Preferably each sector comprises a row or column ofwells. According to a preferred embodiment, the sectors comprise a 1×12array of equal size linear sectors, wherein each sector corresponds to arow of wells.

Preferably, the substrate further comprises: (i) first conductivethrough-holes electrically connecting the working contact surfaces onthe bottom surface with the one or more corresponding patterned workingelectrodes on the top surface and (ii) second conductive through-holeselectrically connecting the counter contact surfaces to the one or morecorresponding patterned counter electrodes on the top surface.Preferably, the one or more working contacts surfaces comprising one ormore circular configurations and the one or more counter contactsurfaces have an elongated configuration, said counter contact surfaces,preferably, being common to more than one, or more preferably, all ofthe sectors.

Another embodiment of the invention, shown in FIG. 10A, relates tomulti-well plate or multi-well plate bottom comprising:

-   -   (a) a substrate having a top surface and a bottom surface, the        plate bottom having an array of regions corresponding to a        standard 96-well plate configuration, the array comprising rows        A, B, C, D, E, F, G, and H and columns 1, 2, 3, 4, 5, 6, 7, 8,        9, 10, 11, and 12;    -   (b) a plurality of working electrodes (preferably patterned) on        the top surface;    -   (c) a plurality of counter electrodes (preferably patterned) on        the top surface, each of the counter electrodes being associated        with corresponding working electrodes; and    -   (d) two or more independently addressable sectors, each sector        having two or more independently addressable contacts on the        bottom surface, the contacts corresponding to one or more        electrodes within assay regions within one of the sectors,

wherein the contacts include first sector contacts comprising:

-   -   (i) one or more working contacts located within one or more        working contact regions at A1-B1, C1-D1, E1-F1 and G1-H1, the        one or more working contacts being electrically connected to the        one or more corresponding, preferably patterned, counter        electrodes on the top surface; and    -   (ii) one or more counter contacts located within one or more        counter contact regions at B1-C1, D1-E1, and F1-G1, the counter        contact surfaces being electrically connected to the one or more        corresponding, preferably patterned, counter electrodes on the        top surface;

wherein the one or more working contacts and the one or more countercontacts are electrically isolated from each other.

Preferably, the electrode surfaces are on the top surface of a platebottom or substrate and the contacts are on the bottom surface, in whichcase the plate substrate advantageously further comprises one or moreconductive through-holes (for example, a hole that is filled or coatedwith a conducting material) electrically connecting the one or moreworking electrode surfaces and the one or more counter electrodesurfaces on the top side with the conductive contacts on the bottomside. Preferably, the plate substrate comprises two or more conductivethrough-holes, more preferably 6 or more, even more preferably 12 ormore and most preferred 24 or more conductive through-holes.

Thus, another embodiment of the invention relates to a multi-well platehaving a plurality of wells comprising a substrate having a top surfaceand a bottom surface, the top surface comprising a plurality ofelectrodes and the bottom surface comprises one or more electricalcontacts, wherein the substrate further includes one or more conductivethrough-holes electrically connecting the one or more electricalcontacts with the electrodes. Preferably, the one or more conductivethrough-holes are located between and/or adjacent the wells rather thandirectly beneath the wells to reduce the likelihood of detrimentalleakage.

Preferably, the substrate includes one or more redundant conductivethrough-holes electrically connected to each of the working electrodesurfaces and the counter electrode surfaces. That is, the workingelectrodes and/or counter electrodes are electrically connected to theplate contacts via two or more through-holes per electrode. Even thougha single through-hole may be sufficient to electrically connect anelectrode to an electrical contact, providing redundant through-holesallows for more uniform distribution of voltage or current.

Another embodiment of the invention relates to a multi-well plate havinga standard 96-well plate configuration, the array comprising one ormore, preferably two or more, more preferably all, of the followingsectors:

-   -   a first sector comprising wells A1 through A4, B1 through B4, C1        through C4, and D1 though D4;    -   a second sector comprising wells A5 through A8, B5 through B8,        C5 through C8, and D5 though D8;    -   a third sector comprising wells A9 through A12, B9 through B12,        C9 through C12, and D9 through D12;    -   a fourth sector comprising wells E1 through E4, F1 through F4,        G1 through G4, and H1 though H4;    -   a fifth sector comprising wells E5 through E8, F5 through F8, G5        through G8, and H5 though H8; and    -   a sixth sector comprising wells E9 through E12, F9 through F1,        G9 through G12, and H9 though H12;    -   the top surface comprising a plurality of electrodes and each        sector comprising one or more sector electrodes;    -   the bottom surface comprising one or more independently        addressable contact sectors on the bottom surface and each of        the contact sectors corresponding to the sector electrodes in a        sector of the plate;

wherein the sectors each further include one or more conductivethrough-boles electrically connecting the one or more contact sectorswith the corresponding sector electrodes, each of the one or moreconductive through-holes being located between the wells and/or adjacentthe wells.

Preferably, each sector comprises two, more preferably three, even morepreferably four and most preferred at least eight through-holes.Preferably, each sector comprises eight through-holes in a 2×4 array.According to one preferred embodiment, the through-boles comprisethrough-hole pairs.

According to one preferred embodiment, the through-holes comprise:

-   -   (i) two or more, preferably three or more, more preferably all        of first sector through-holes comprising four through-holes        along sector edge adjacent A1-A4 and four through-holes at        C1-D1; C2-D2; C3-D3 and C4-D4;    -   (ii) two or more, preferably three or more, more preferably all        of second sector through-holes comprising four through-holes        along sector edge adjacent A5-A8 and four through-holes at        C5-D5; C6-D6; C7-D7 and C8-D8;    -   (iii) two or more, preferably three or more, more preferably all        of third sector through-holes comprising four through-holes        along sector edge adjacent A9-A12 and four through-holes at        C9-D9; C10-D10; C11-D11 and C12-D12;    -   (iv) two or more, preferably three or more, more preferably all        of fourth sector through-holes comprising four through-holes        along sector edge adjacent H1-H4 and four through-holes at        E1-F1; E2-F2; B3-F3 and E4-F4;    -   (v) two or more, preferably three or more, more preferably all        of fifth sector through-holes comprising four through-holes        along sector edge adjacent H5-H8 and four through-holes at        E5-F5; E6-F6; E7-F7 and E8-F8; and    -   (vi) two or more, preferably three or more, more preferably all        of sixth sector through-holes comprising four through-holes        along sector edge adjacent H9-H12 and four through-holes at        E9-F9; E10-F10; E11-F11 and E12-F12.

Preferably, each of the sector through-holes located within the firstconductive region of the sectors electrically connects one or morecontacts on the bottom surface to one or more working electrodes on thetop surface and/or each of the sector through-holes located within thesecond conductive region of the sectors electrically connects one ormore contacts on the bottom surface to one or more counter electrodes onthe top surface.

Yet another embodiment of the invention relates to a multi-well platehaving a standard 384-well plate configuration, the array comprisingrows A through P and columns 1 through 24, the array comprising one ormore, preferably two or more, more preferably four or more and mostpreferred six of the following:

-   -   a first sector comprising wells A1 through A8, B1 through B8, C1        through C8, D1 though D8, E1 through E8, F1 through F8, G1        through G8, and H1 though H8;    -   a second sector comprising wells A9 through A16, B9 through B16,        C9 through C16, D9 though D16, E9 through E16, F9 through F16,        G9 through G16, and H9 though H16;    -   a third sector comprising wells A17 through A24, B17 through        B24, C17 through C24, D17 though D24, E17 through E24, F17        through F24, G17 through G24, and H17 though H24;    -   a fourth sector comprising wells I1 through I8, J1 through J8,        K1 through K8, L1 though L8, M1 through M8, N1 through N8, O1        through O8 and P1 through P8;    -   a fifth sector comprising wells I9 through I16, J9 through J16,        K9 through K16, L9 though L16, M9 through M16, N9 through N16,        O9 through O16 and P9 through P16; and    -   a sixth sector comprising wells I17 through I24, J17 through        J24, K17 through K24, L17 though L24, M17 through M24, N17        through N24, O17 through O24 and P17 through P24;

a top surface comprising a plurality of electrodes and each sectorcomprising one or more sector electrodes; and

a bottom surface comprising one or more independently addressablecontact sectors on the bottom surface and each of the contact sectorscorresponding to the sector electrodes;

wherein the sectors each further include one or more conductivethrough-holes electrically connecting the one or more contact sectorswith the corresponding sector electrodes, the one or more conductivethrough-holes being located between the wells and/or adjacent to thewells.

Preferably, each sector comprises at least two, preferably at leastthree, more preferably at least four and even more preferably at leasteight through-holes. According to a preferred embodiment, each sectorcomprises sixteen through-holes, preferably arranged in a 2×8 array.Preferably, the through-holes comprise through-hole pairs.

According to another preferred embodiment, the through-holes comprise:

-   -   (i) two or more, preferably three or more, more preferably all        of first sector through-holes comprising four through-holes        along sector edge adjacent A1-A8 and four through-holes along        G1-H1 through G8-H8;    -   (ii) two or more, preferably three or more, more preferably all        of second sector through-holes comprising four through-holes        along sector edge adjacent A9-A16 and four through-holes along        G9-H9 through G16-H16;    -   (iii) two or more, preferably three or more, more preferably all        of third sector through-holes comprising four through-holes        along sector edge adjacent A17-A24 and four through-holes along        G17-H17 through G24-H24;    -   (iv) two or more, preferably three or more, more preferably all        of fourth sector through-holes comprising four through-holes        along sector edge adjacent P1-P8 and four through-holes along        I1-J1 through I8-J8;    -   (v) two or more, preferably three or more, more preferably all        of fifth sector through-holes comprising four through-holes        along sector edge adjacent P9-P16 and four through-holes along        I9-J9 through I16-J16; and    -   (vi) two or more, preferably three or more, more preferably all        of sixth sector through-holes comprising four through-holes        along sector edge adjacent P-17 through P-24 and four        through-holes along I17-J17 through I24-J24.jbjb

5.2 Embodiments of Multi-Well Assay Plates of the Invention

In the following sections a variety of embodiments of assay modules,particularly multi-well assay plates of the invention are described. Thefigures will show plates having specific numbers and arrangements ofwells, typically the figures show 96-well plates having a 12×8 array ofwells. The description of the structure and the elements of the plates,however, are understood to be generic in the sense that they can applyor be readily adapted to a variety of assay modules including plateshaving any arbitrary number of wells in any arbitrary arrangement (e.g.,any of the standard plate formats used in high-throughput screening).

5.2.1 Multi-Layer Electrode Plates

FIG. 5 shows an example of a multi-well assay plate of the invention.Multi-well assay plate 500 comprises a laminar structure comprising, insequence, a first conductive layer 508, a dielectric layer 506, a secondconductive layer 504 and a plate top 502. Holes 503 through plate top502, holes 505 through second conductive layer 504, and holes 507through dielectric layer 506 (the holes having interior surfaces 509,510 and 512, respectively) are aligned so as to form a plurality ofwells having well bottoms defined by first conductive layer 508 and wellwalls defined by the interior surfaces 509, 510 and 512. Thecross-sectional shape of the holes in the plane of the laminar structuremay be circular, square or any arbitrary shape. The interior walls ofthe holes may be perpendicular to the plane of the laminar structure soas to provide cylindrical wells or they may be shaped, e.g., to giveconical or hemispherical wells. In one embodiment of the invention, thediameters of the holes 503, 505 and 507 are the same; in thisembodiment, only the interior surfaces 510 of second conductive layer504 are exposed to the volumes of the wells of the plate. Alternatively,the diameters of the holes 503 may be larger than the diameters of holes505 and 507 so as to expose some of the top surface of conductive layer504 to the volumes of the wells of the plate.

First conductive layer 508 is a material suitable for use as a counterelectrode or, preferably, a working electrode in an ECL assay (seedescription of ECL electrodes above). In one embodiment it is aconductive sheet of material such as a metal sheet or foil (e.g.,platinum or gold foil) or a sheet of conductive plastic. Preferably, itis a sheet of conductive plastic composite comprising carbon particles(e.g., carbon fibrils) dispersed in a polymeric matrix. In an alternateembodiment, conductive layer 508 is a film of a conductive materialsupported on a substrate. Suitable films include coatings such asconducting inks comprising conducting particles dispersed in a matrix(e.g., carbon or metal-based conducting inks) or metal or carbon films(e.g., metal or carbon films deposited on a substrate via evaporative orCVD processes or lamination). Suitable substrates include plastic sheet,glass and ceramic. Electrical contact to first conductive layer 508 canbe made by contacting any exposed conductive surface, preferably, thebottom surface. Electrical contact to the top of first conductive layer508 can be facilitated by extending the width and/or length of the layerbeyond that of the other layers. In the embodiment where firstconductive layer 508 comprises a conductive coating on a non-conductivesubstrate, electrical connection may be made to the bottom of the plateby incorporating through-holes through the non-conductive substrate.Such through-holes are, preferably, made conductive by inserting aconductive material such as a metal wire or by filling with a conductivematerial such as a metal-filled ink so as to provide a high conductivitypath from the conductive coating to the bottom of the plate. Accordingto another embodiment, the holes may be filled with carbon-filled ink.Typically, conductive layer 508 provides a fluid impermeable barrier andacts to contain fluid held within the wells. However, a porousconductive layer 508 may be, optionally, employed to conduct dot-blotassays and other assays that benefit from filtration of samples orreagents through the bottom of the plate and/or working electrode.

Dielectric layer 506 is an electrically insulating material and preventsconductive layers 504 and 508 from coming into electrical contact.Suitable materials include sheets of non-conductive plastic, glass orceramic, preferably comprising an adhesive coating on one or both sidesso as to provide adhesive bonds to conductive layers 504 and/or 508(e.g., single or double sided adhesive tape). In such dielectric layers,holes 507 may be formed by a molding process (i.e., during fabricationof the layer), by selective etching or, preferably, through a cuttingprocess conducted prior to final assembly of the plate, e.g., by diecutting or laser drilling. Alternatively, dielectric layer 506 is anelectrically insulating coating such as a dielectric ink, a polymericfilm, a photoresist film, and/or a ceramic or glass film (e.g., ceramicor glass films deposited by evaporative or CVD processes). Holes 507 insuch layers may be formed by cutting, by a selective etching of thecoating (e.g., by photolithography or by etching in the presence of aphysical mask) or by a patterned deposition of the coating via a processlike screen printing, laser printing or ink jet printing or depositionthrough a mask.

Second conductive layer 504 is a material suitable for use as a workingelectrode or, preferably, a counter electrode in an ECL assay (seedescription of ECL electrodes above). Optionally, second conductivelayer 504 may be omitted (e.g., when plate top 502 comprises anelectrode material or when the apparatus used to analyze the plate iscapable of supplying an electrode (e.g., in the form of one or moreconductive probes). In one embodiment it is a conductive sheet ofmaterial such as a metal sheet or foil (e.g., aluminum, platinum or goldfoil) or a sheet of conductive plastic (e.g., a sheet of conductiveplastic composite comprising carbon particles dispersed in a polymericmatrix). In such conductive layers, holes 505 may be formed by a moldingprocess (i.e., during fabrication of the layer), by selective etchingor, preferably, through a cutting process conducted prior to finalassembly of the plate, e.g., by die cutting or laser drilling. In analternate embodiment, conductive layer 504 is a film of a conductivematerial supported on a substrate. Suitable films include coatings suchas conducting inks comprising conducting particles dispersed in a matrix(e.g., carbon or metal-based conducting inks) or metal films (e.g.,metal films such as aluminum, gold and platinum deposited on a substratevia evaporative or CVD processes). Suitable substrates include plasticsheet, glass and ceramic. Holes 505 in such layers may be formed bycutting, by selective etching of the coating (e.g., by photolithographyor by etching in the presence of a physical mask) or by patterneddeposition of the coating via a process like screen printing, laserprinting or ink jet printing or deposition through a mask. In apreferred embodiment of the invention, dielectric layer 506 and secondconducting layer 504 are both provided by the layers of a metal-coatedadhesive tape (i.e., a laminar structure comprising a layer of adhesiveadjacent to a non-conducting plastic sheet that is coated on theopposite side with a metallic film (preferably an evaporated film ofaluminum). Preferably, holes 505 and 507 are simultaneously formed insuch a tape by a cutting process such as die cutting or laser drillingprior to assembly of the plate. Electrical contact to second conductivelayer 504 can be made by contacting any exposed conductive surface.Electrical contact to the top or bottom of second conductive layer 504can be facilitated by extending the width and/or length of the layerbeyond that of the plate top. Alternatively, openings through the otherlayers can be incorporated to provide for additional exposed surfaces ofsecond conductive layer 504. Such openings can be through-holes madeconductive by inserting a conductive material such as a metal wire or byfilling with a conductive material such as a metal-filled ink so as toprovide a high conductivity path from second conductive layer 504 to thebottom or top of the plate. Optionally, multi-well assay plate 500comprises an additional conductive layer (not shown) that comprises amaterial suitable for use as a reference electrode. This referenceelectrode layer should be electrically isolated from the otherconductive layers, e.g., through the use of additional dielectric layersas necessary.

Plate top 502 is a plate top as described earlier in the application.Preferably, it complies with industry standards for microplatedimensions and well number so as to be compatible with commerciallyavailable equipment for storing, moving and processing microplates.Plate top 502 is generally made of a non-conductive plastic. It may bemade of a conducting material or coated with a conductive material(suitable for acting as a working electrode or, preferably, a counterelectrode in an ECL assay) in which case, second conductive layer 504may be omitted. Preferably, the bottom surface of plate top 502comprises an adhesive coating so as to provide adhesive bonding tosecond conductive layer 504. In some alternate embodiments, plate top502 is omitted.

In use, components 502, 504, 506 and 508 of plate 500 should be sealedagainst adjoining layers so as to prevent the leakage of fluidscontained within the wells of plate 500. Sealing may be accomplished byphysically holding the components together under pressure through theuse of fasteners and/or clamps. Such fasteners and/or clamps may beintegrated into plate 500 and/or they may be comprised in an externalfixture. Alternatively, sealing may be accomplished through the use ofadhesive coatings on the surfaces of the components. In some cases, thecomponents may have inherently adhesive properties; for example,evaporated films, CVD films, cast polymer films, printed inks, etc. canbe designed to adhere to the substrate on which they are deposited. Someof the seals may also be accomplished through a welding process such asultrasonic welding or solvent welding (i.e., by applying a solvent thatsoftens or partially dissolves one or both of the surfaces to be sealedtogether). The layers of the plate are, preferably, aligned so that theexposed area of conductive layer 508 is centered in the well andsurrounded by the exposed area of conductive layer 504.

In operation, test samples are introduced into wells of plate 500. Asource of electrical energy is connected across first and secondconducting layers 508 and 504. Application of electrical energy acrossthese connections leads to the application of an electrochemicalpotential across the test samples via the exposed surfaces of conductinglayers 508 and 504. In the case of an ECL assay, it is preferable toapply electrical energy so as to generate ECL at or near the surface ofconductive layer 508 (i.e., conductive layer 508 provides the workingelectrode and conductive layer 504 provides the counter electrode) sothat light is generated near the center of the well.

In some preferred embodiments, the plates are divided into individuallyaddressable sectors of jointly addressable wells. Such sectoring may beaccomplished by dividing conductive layers 504 and/or 508 into aplurality of individually addressable sections. FIG. 6A shows a secondconductive layer 600 analogous to second conductive layer 504 describedabove except that the layer is sectioned into six square sections(602A-F) that are electrically isolated from each other. Applying apotential to, e.g., section 602A, will result in the potential beingselectively applied to fluid in the plate sector defined by the wells incontact with section 602A. Such sectoring allows for the sequentialinduction and measurement of ECL from each sector in the plate. Somealternate sectioning schemes are illustrated in FIG. 6B (conductivelayer 620 is sectioned into columnar sectors 622A through 622L) and FIG.6C (conductive layer 640 is sectioned into individual well sectors 644).FIG. 7A shows a first conductive layer 700 analogous to first conductivelayer 508 described above except that the layer is sectioned into sixsquare sections (702A-F) that are electrically isolated from each other.Applying a potential to, e.g., section 702A, will result is thepotential being selectively applied to fluid in the plate sector definedby the wells in contact with section 702A. Some alternate sectioningschemes are illustrated in FIG. 7B (conductive layer 720 is sectionedinto columnar sectors 722 along the width of the plate), FIG. 7C(conductive layer 740 is sectioned into columnar sectors 742 along thelength of the plate), and FIG. 7D (conductive layer 760 is sectionedinto individual well sectors 766). In general, to divide plate 500 intoindependently addressable sectors, it is only required that one ofconductive layers 504 and 508 of plate 500 be sectioned since electricalconnections must be made to both conductive layers in contact with aspecific well in order to complete an electrochemical circuit. In someembodiments, sectioning of both conductive layers 504 and 508 is used tomaximize the number of individually addressable sectors while minimizingthe number of electrical contacts. For example, a plate formed usingfirst conductive layer 740 (as shown in FIG. 7C) with second conductivelayer 620 (as shown in FIG. 6B) allows for the individual addressing of96 wells while requiring only 20 electrical contacts. Sectioning ofconductive layers 504 and/or 508 of plate 500 may be achieved by avariety of methods including cutting processes such as die cutting ordrilling, selective etching, such as by photolithography or by etchingthrough a mask, or by selective deposition.

5.2.2 Single Electrode Layer Plates

Sectioning of a conductive layer may be used to provide multipleindependent electrodes within a given well. FIG. 16A shows anotherexample of a multi-well assay plate of the invention. Multi-well assayplate 1600 is similar in structure to multi-well assay plate 500 fromFIG. 5 except that it has a single conductive layer on a support, theconductive layer being sectioned so as to provide two or moreindependent electrodes (e.g., a counter and a working electrode) in agiven well. Multi-well assay plate 1600 is a laminar structurecomprising in sequence a plate top 1602, an adhesive layer 1604, adielectric layer 1606, a conductive layer 1608 and a substrate 1610.Holes 1603 and 1605 through plate top 1602 and adhesive layer 1604,respectively, form a plurality of wells having well bottoms defined bydielectric layer 1606, conductive layer 1608 and/or substrate 1610.Conductive layer 1608 is sectioned into two electrically isolatedsections, a working electrode section 1620 and a counter electrodesection 1622. The sectioning is designed so that a fluid in a given wellis exposed to surfaces of both sections. Element 1612 shows layers 1606,1608, and 1610 aligned and stacked, in order from top to bottom—1606(top), 1608 and 1610 (bottom)—so as to form a plate bottom withintegrated electrodes.

Plate top 1602 is a plate top as described earlier in the application.Preferably, it complies with industry standards for microplatedimensions and well number so as to be compatible with commerciallyavailable equipment for storing, moving and processing microplates.Plate top 1602 is generally made of a non-conductive plastic. Adhesive1604 is preferably an adhesive coating or adhesive tape suitable forsealing plate top 1602 to element 1612 and preferably forms fluid tight,high resistance seals. Adhesive 1604 is, preferably, a double sidedadhesive tape. Optionally, adhesive 1604 may be omitted. In such case,plate top 1602 may be sealed to element 1612 via an adhesive coating onplate top 1602 or via other sealing techniques such as heat sealing,solvent welding, sonic welding or through the use of applied pressure byclamping. In some alternate embodiments, plate top 1602 is omitted.

Dielectric layer 1606 is an electrically insulating material and servesto define the regions of conductive layer 1608 that contact fluid in awell. In FIG. 16A, dielectric 1606 covers all of working electrodesection 1620 that is exposed to the volume of a given well except for aregion defined by holes 1607 in dielectric layer 1606. The boundariesformed by holes 1607 define a fluid containment region over workingelectrode 1620 that can be used to confine a small volume of fluid incontact with the exposed electrode but not in contact with the otherexposed surfaces within the well. Such fluid containment regions may beused, advantageously, for selectively immobilizing a reagent on theactive area of the working electrode in a well. Alternatively,dielectric layer 1606 may be omitted. Preferably, dielectric layer 1606is an electrically insulating coating such as a dielectric ink, apolymeric film, a photoresist film, and/or a ceramic or glass film(e.g., ceramic or glass films deposited by evaporative or CVDprocesses). Holes 1607 in such layers may be formed by selective etchingof the coating (e.g., by photolithography or by etching in the presenceof a physical mask) or patterned deposition of the coating via a processlike screen printing, laser printing or ink jet printing or depositionthrough a mask. Alternatively, dielectric 1606 may be a sheet ofnon-conductive plastic, glass or ceramic, preferably comprising anadhesive coating on one or both sides so as to provide adhesive bonds toadjoining layers. In such dielectric layers, holes 1607 may be formed bya molding process (i.e., during fabrication of the layer), by selectiveetching or, preferably, through a cutting process conducted prior tofinal assembly of the plate, e.g., by die cutting or laser drilling.

Conductive layer 1608 comprises materials suitable for use as counterelectrodes or working electrodes in an ECL assay (see description of ECLelectrodes above). Working electrode section 1620 and counter electrodesection 1622 may comprise different materials (so as to optimize eachfor its function) or they may comprise the same materials (so as tosimplify their formation, e.g., by printing both sections in one screenprinting step). Preferably, conductive layer 1608 is a coating of aconductive material supported on a substrate 1610. Suitable filmsinclude coatings such as conducting inks that comprise conductingparticles dispersed in a matrix (e.g., carbon or metal-based conductinginks) or metal films (e.g., metal films deposited on a substrate viaevaporative or CVD processes). Formation of the patterned sections maybe accomplished by selective patterned deposition of the film (e.g., byscreen printing, laser printing, ink jet printing, evaporation through amask, etc.) and/or by selective patterned etching of a contiguous film(e.g., by photolithographic and chemical etching protocols used in thesemiconductor industry). Alternatively, sections 1620 and 1622 aresections cut, e.g., by die cutting, from a conductive sheet of materialsuch as a metal sheet or foil (e.g., platinum, gold, steel, or aluminumfoil) or a sheet of conductive plastic, such as a sheet of conductiveplastic composite comprising carbon particles (e.g., carbon fibrils)dispersed in a polymeric matrix. Optionally, conductive layer 1608comprises an additional section (not shown) that comprises a materialsuitable for use as a reference electrode.

Substrate 1610 is a non-conductive material such as plastic sheet, glassor ceramic. Electrical contact to electrode sections 1620 and 1622 maybe made to any exposed surface. Electrical contact to the top of theelectrode sections can be facilitated by extending the width and/orlength of the sections (as well as that of substrate 1610) beyond thatof plate top 1602. Alternatively, electrical connections may be made tothe bottom of the plate by incorporating through-holes through substrate1610. Such through-holes are, preferably, made conductive by inserting aconductive material such as a metal wire or by filling with a conductivematerial such as a metal-filled ink so as to provide a high conductivitypath from the electrode sections to the bottom of the plate.

In use, components 1602, 1606 and 1608 of plate 1600 should be sealedagainst adjoining layers so as to prevent the leakage of fluidscontained within the wells of plate 1600. Sealing may be accomplished byphysically holding the components together under pressure through theuse of fasteners and/or clamps. Such fasteners and/or clamps may beintegrated into plate 1600 and/or they may be comprised in an externalfixture. Alternatively, sealing may be accomplished through the use ofadhesive coatings on the surfaces of the components (see discussionabove in Section 5.1). Suitable adhesives include acrylic adhesives (3M200MP).

According to one embodiment, polypropylene plate tops are used. In orderto attach the plate tops to the bottoms, it is preferably to use a lowsurface energy (LSE) adhesive. The only difference between the LSEadhesive and a non-LSE adhesive is the flow or “wet-out”characteristics. Non-LSE adhesives such as 3M 200MP will not stick aswell to surfaces characterized as having a low surface energy such aspolypropylene. One suitable adhesive for use with polypropylene is amodified acrylic such as 3M 300LSE, which is designed specifically forlow surface energy plastics.

In some cases, the components may have inherently adhesive properties;for example, evaporated films, CVD films, cast polymer films, printedinks, etc. can be designed to adhere to the substrate on which they aredeposited. Some of the seals may also be accomplished through a weldingprocess such as sonic welding or solvent welding (i.e., by applying asolvent that softens or partially dissolves one or both of the surfacesto be sealed together, contacting the two surfaces and then allowing thesurfaces to re-harden to form a bond). The layers of the plate are,preferably, aligned so that the exposed area of section 1620 is centeredin the well and surrounded on two sides by the exposed area of section1622.

In operation, test samples are introduced into wells of plate 1600. Asource of electrical energy is connected across working electrodesection 1620 and counter electrode section 1622. Application ofelectrical energy across these connections leads to the application ofan electrochemical potential across the test samples via the exposedsurfaces of working electrode section 1620 and counter electrode section1622.

In some preferred embodiments, the plates are divided into individuallyaddressable sectors of jointly addressable wells. Such sectoring may beaccomplished by further dividing working electrode section 1620 and/orcounter electrode section 1622 into a plurality of individuallyaddressable sections. FIG. 16B shows a conductive layer 1640 having aworking electrode section 1642 and counter electrode section 1644.Conductive layer 1640 is analogous to conductive layer 1608 describedabove except that working electrode section 1642 is further divided into12 subsections (1642A-L) that are electrically isolated from each other.Applying a potential to, e.g., subsection 1642A, will result in thepotential being selectively applied to fluid in the plate sector definedby the wells in contact with subsection 1642A. An alternate sectioningscheme is illustrated in FIG. 16C (sectioning of working electrodesection 1662 and counter electrode section 1664 of conductive layer 1660into six square sectors). In general, to divide plate 1600 intoindependently addressable sectors, it is only required that one ofworking electrode section 1620 and counter electrode section 1622 befurther sectioned since electrical connections must be made to bothconductive layers in contact with a specific well in order to completean electrochemical circuit. In some embodiments, the patterning of thesections is simplified by subsectioning both working electrode section1620 and counter electrode section 1622.

5.2.3 Specific Embodiments of Multi-Layer Electrode Plates

Another aspect of the invention relates to assay plates, preferablymulti-well plates, wherein one or more electrode surfaces are formedusing conductive foils or conductive films or layers which are adjoinedto form the assay plate.

One embodiment of the invention relates to a multi-well assay plate forconducting assays comprising:

-   -   (a) a first electrically conductive layer, preferably        partitioned into two or more electrically isolated sectors;    -   (b) an insulating layer having a plurality of insulating layer        openings;    -   (c) a second electrically conductive layer on the insulating        layer, the second electrically conductive layer having a        plurality of second electrically conductive layer openings, the        second layer preferably being partitioned into two or more        electrically isolated sectors; and    -   (d) a plate top having a plurality of plate top openings;

wherein the insulating layer is between the first electricallyconductive layer and the plate top and wherein the insulating layeropenings, the second electrically conductive layer openings and theplate top openings are aligned forming a plurality of wells.

According to another embodiment, the first conductive layer comprises asubstrate comprising partitioned electrically conductive surfaces.Preferably, the layer is “partitioned” to correspond or align with aplate sector.

According to another embodiment, the first conductive layer comprises asubstrate comprising an electrically conductive surface and the secondlayer comprises a partitioned conductive film on the insulating layerhaving a plurality of conductive film openings.

Preferably, the first conductive layer comprises a fibril composite witha bottom coated with or painted with a conductive metal, preferablysilver.

A still further embodiment relates to a multi-well plate for conductingassays (preferably electrode induced luminescence, more preferablyelectrochemiluminescence assays) comprising:

-   -   (a) an electrically conductive layer,    -   (b) an insulating layer having a plurality of insulating layer        openings;    -   (c) a conductive film on the insulating layer having a plurality        of conductive film openings, the conductive film being        partitioned into two or more electrically isolated sectors; and    -   (d) a plate top having a plurality of plate top openings;        wherein the insulating layer is between the electrically        conductive layer and plate top and the insulating layer openings        and the plate top openings are aligned forming wells for        conducting the assays.

Another aspect relates to a multi-well plate for conducting assays(preferably electrode induced luminescence, more preferablyelectrochemiluminescence assays) comprising:

-   -   (a) an electrically conductive layer partitioned into two or        more electrically isolated sectors;    -   (b) an insulating layer having a plurality of insulating layer        openings;    -   (c) a conductive film on the insulating layer having a plurality        of conductive film openings, the conductive film being        partitioned into two or more electrically isolated sectors; and    -   (d) a plate top having a plurality of plate top openings;

wherein the insulating layer is between the electrically conductivelayer and plate top and the insulating layer openings and the plate topopenings are aligned forming wells for conducting the assays.

FIG. 8B shows an exploded view of multi-well assay plate 830, anembodiment of plate 500 (shown in FIG. 5) that comprises additionaladaptations allowing for convenient electrical connections to theconductive layers of the plate. FIG. 8C shows a stylized cross-sectionalview of two wells 842A and 842B of plate 830. Plate 830 comprises alaminar structure comprising, in sequence, plate top 832, adhesive layer844, conductive tape layer 852B, conductive layer 858, and conductivetape layer 852A. Conductive tape layers 852A and 852B are provided byfolding conductive tape 848 around conductive layer 858 at fold 854.Holes 834, 846, and 856 through plate top 832, adhesive layer 844, andconductive tape layer 852B, respectively, are aligned so as to form aplurality of wells having well bottoms defined by conductive layer 858.A plurality of holes 850 through conductive tape layer 852A allow forelectrical contact to conductive layer 858 from the bottom of plate 830.After folding of conductive tape 848, holes 850 are, preferably, alignedwith regions between the wells of plate 830. For example, FIG. 8C showsa cross-sectional view of two wells (842A and 842B) of plate 830. Hole853 through conductive tape layer 852A exposes the bottom surface ofconductive layer 858 to provide electrical contact 872. Contact 872 islocated on the region of conductive layer 858 between wells 842A and842B; this arrangement allows an electrical connection to be made tocontact 872 without distorting or disturbing the surface of conductivelayer 858 exposed to wells 842A or 842B.

Plate top 830 is a plate top analogous to plate top 502 in FIG. 5.Adhesive layer 844 is an adhesive suitable for forming a fluid-tightseal between plate top 502 and conductive tape layer 852B. Adhesivelayer 844 may be an adhesive coating applied, e.g., by spray coating,onto plate top 502 or conductive tape layer 852B. In a preferredembodiment, adhesive layer 844 is a double sided adhesive tape (i.e. aplastic film coated on both sides with adhesive). Holes 846 arepreferably formed by a cutting process such as laser drilling or diecutting. Conductive tape 848 is preferably a laminar structurecomprising conductive film 864, dielectric film 866 and adhesive film868. Conductive film 864 is a material suitable for use as a workingelectrode or, preferably, a counter electrode in an ECL assay (see forexample the description of the analogous second conductive layer 504 inFIG. 5). Optionally, conductive film 864 may be omitted (e.g., whenplate top 832 comprises an electrode material or when the apparatus usedto analyze the plate is capable of supplying an electrode (e.g., in theform of one or more conductive probes). Conductive film 864 ispreferably of sufficient conductivity so that, during use of the platein an assay, a potential applied to conductive film 864 in conductivetape layer 852A will be evenly distributed over the surface ofconductive film 864 in conductive tape layer 852B. Dielectric film 866is an electrically insulating film suitable for preventing electricalcontact between conductive film 864 and conductive layer 858. Adhesivefilm 868 is an adhesive suitable for forming a fluid-tight seal betweendielectric film 866 and conductive layer 858. Optionally, adhesive film868 provides for electrical isolation of conductive film 864 andconductive layer 858; dielectric film 866 may then be omitted. In oneembodiment, conductive tape 848 is an electrically insulating plasticfilm coated on one side with an adhesive coating and on the other sidewith a conductive coating such as an evaporated metal film, preferablycomposed of aluminum. Holes 850 and 856 are preferably formed by acutting process such as laser drilling or die cutting.

Conductive layer 858 is a material suitable for use as a counterelectrode, or preferably a working electrode in an electrode inducedluminescence assay (see for example the description of the analogousfirst conductive layers 508 in FIGS. 5 and 700, 720, 740 and 760 inFIGS. 7A-7D). Preferably, conductive layer 858 is a composite comprisingcarbon particles, most preferably carbon fibrils, distributed in apolymeric matrix. It is preferably sectioned into 12 electricallyisolated columnar sections corresponding to a column of wells in plate830. Holes 850 in conductive tape layer 852A expose the bottom surfaceof conducting layer 858. It is desirable that a potential applied to thebottom of conductive layer 858 leads to an even distribution ofpotential over the regions of the top surface of conductive layer 858that form the bottom of the wells in plate 830. Such even distributionof potential may be achieved by providing multiple evenly-distributedsites for making electrical connection to conductive layer 858. Forexample, holes 850 are arranged are arranged in a 4.times.12 array so asto expose the bottom of conductive layer 858 in the regions centeredbetween the first and second wells, the third and fourth wells, thefifth and sixth wells, and the seventh and eighth wells of the columnsof wells of plate 830. Even distribution of potential is also aided bythe conductivity of conductive layer 858. When a material of onlymoderate conductivity is used, e.g., a composite of carbon particlesdistributed in a matrix, it is advantageous that conductive layer 858comprise a highly conductive coating on the bottom of the layer so as tobetter distribute potential across the surface of the layer. Preferredhighly conductive coatings include metal films (e.g., evaporated,electro-deposited or electroless-deposited films) and metal-containingpaints (e.g., silver paint). Suitable silver paints include materialsavailable from E. I. du Pont de Nemours and Co. (e.g., Dupont 5000,5007, 5008, 5021, 5025, 5028, 5031, and 5089), Acheson Colloids Co.(e.g., Acheson PD-020, 479SS, 478SS, 725A, PF-007, EL-010, 820C, andElectrodag 506SS), and Conductive Compounds Inc. (e.g., AG-410 andAG-500).

In operation, test samples are introduced into wells of plate 830. Asource of electrical energy is connected to conducting film 864 and oneor more sections of conductive layer 858. Preferably, the connection toconducting film 864 is made by contacting conductive tape layer 852A onthe bottom of the plate. Preferably, the connection to the one or moresections of conductive layer 858 is made by contacting the bottom ofconductive layer 858 via holes 850 in conductive tape layer 852A.Application of electrical energy across these connections leads to theapplication of an electrochemical potential across the test samples viathe exposed surfaces of conducting film 864 and conductive layer 858(the application of electrochemical potential being confined to wells insectors contacting the one or more sections of conductive layer 858). Inthe case of an ECL assay, it is preferable to apply electrical energy soas to generate ECL at or near the surface of conductive layer 858.(i.e., conductive layer 858 provides the working electrode) so thatlight is generated near the center of the well.

FIG. 8B shows a multi-well assay plate with an 8×12 array of wellsdivided into columnar sectors via sectioning of conductive layer 858. Itshould be readily apparent that the design is readily adaptable toplates having different numbers of wells, different arrangements ofwells or different arrangements of sectors (e.g., see discussion of 1536plate below). FIG. 8A shows an alternative sectoring scheme. Multi-wellassay plate 800 comprises a laminar structure comprising, in sequence,plate top 804, adhesive layer 806, conductive tape 810 providingconductive tape layer 814B, and conductive layer 820. Conductive tape810 is folded at fold 818 to provide conductive tape layer 814A adjacentto conductive layer 820 (and on the opposite side of conductive layer820 from conductive tape layer 814B). The components of plate 800 areanalogous to those described for plate 830 except: i) conductive layer820 is sectioned into six square sections (corresponding in a 96-wellplate to six sectors each having a 4×4 array of wells) and ii) thearrangement of holes 812 through conductive tape layer 814A is optimizedfor this arrangement of sectors. Holes 812 are arranged in a 4×6 arraywith each hole centered between the first and second well and betweenthe third and fourth well in each major diagonal defined by each 4×4sector of wells. Plates analogous to plates 800 but having differentnumber of wells, e.g., 384, 1536, or 9600 wells can be made bysubstituting the desired arrangement of holes, preferably using anindustrial standard, in plate top 802, adhesive layer 806 and conductiveplate layer 814B.

Yet another embodiment of the invention relates to a 1536-well platewherein the counter electrode (e.g., see, counter electrode conductivetape 810 of FIG. 8A) wraps around working electrode(s) (e.g., see,working electrode conductive layer 820 of FIG. 8A) with access holes forworking electrode contacts to pass through the counter electrode wrapand make contact with working electrodes (or contacts for the workingelectrodes). Preferably, four working electrode contacts contact theplate bottom (through the holes in the counter electrode wrap) and arearranged in a square to evenly distribute current across the workingelectrode area (e.g., see sectored working electrode surfaces 820 inFIG. 8A). Preferably, two counter electrode contacts make contact withthe counter electrode sheet so as to provide redundancy and protectionfrom a single dirty contact region or connector.

Yet another embodiment relates to a multi-well plate formed by stackingthe conductive and dielectric layers and then drilling holes through themulti-layer composite thereby forming wells, preferably wells havingwalls comprising counterelectrode and/or working electrode surfaces. Forexample, referring to FIG. 5, a multi-well plate (preferably a 1536 orhigher well plate) is formed using a first conductive layer 508, adielectric layer 506, a second conductive layer 504 and a plate top 502,wherein one or more of the layer do not have holes (e.g., holes 502, 505and/or 507). After the layers are bonded together, holes 502, 505 and/or507 may be formed by drilling holes through the respective layers with alaser or the like thereby forming a multi-well plate where each wellcomprises a working electrode surface (e.g., exposed first conductivelayer 508 forming the well bottom) and a counterelectrode surface (e.g.,exposed second conductive layer forming part of the well wall).

5.2.4 Specific Embodiments of Plates Having Conductive Plate Tops

Another aspect of the invention relates to multi-well plates having aplate top coating with a conductive surface (e.g., a painted plate top).The painted plate top can then be affixed to a substrate having aworking electrode surface whereby the conductive surfaces of the platetop may advantageously provide a counter electrode surface.

One embodiment relates to a multi-well plate comprising:

-   -   (a) an electrically conductive layer;    -   (b) an insulating layer having a plurality of insulating layer        openings; and    -   (c) a plate top comprising an electrically conductive surface,        the plate top having a plurality of plate top openings;

wherein the insulating layer is between the electrically conductivelayer and the plate top and the insulating layer openings and the platetop openings are aligned forming wells for conducting assays.

Another embodiment relates to multi-well plate comprising:

-   -   (a) a substrate surface having a plurality of electrodes        patterned thereon;    -   (b) an insulating layer having a plurality of insulating layer        openings; and    -   (c) a plate top comprising an electrically conductive surface,        the plate top having a plurality of plate top openings;

wherein the insulating layer is between the substrate surface and theplate top and the insulating layer openings and the plate top openingsare aligned forming plate openings over one or more of the plurality ofelectrodes thereby forming wells.

A still further embodiment relates to a multi-well plate comprising:

-   -   (a) an electrically conductive layer partitioned into two or        more electrically isolated sectors;    -   (b) an insulating layer having a plurality of insulating layer        openings; and    -   (c) a plate top comprising an electrically conductive surface,        the plate top having a plurality of plate top openings;

wherein the insulating layer is between the electrically conductivelayer and the plate top and the insulating layer openings and the platetop openings are aligned forming wells.

Yet another embodiment relates to a multi-well plate comprising:

-   -   (a) an electrically conductive layer,    -   (b) an insulating layer having a plurality of insulating layer        openings; and    -   (c) a plate top comprising an electrically conductive surface        partitioned into two or more electrically isolated sectors, the        plate top having a plurality of plate top openings;

wherein the insulating layer is between the electrically conductivelayer and the plate top and the insulating layer openings and the platetop openings are aligned forming wells.

In certain embodiments of plate 800 (or 830) (FIGS. 8A and 8B),conductive tape 810 (or 848) may be omitted. In these embodiments, platetop 802 (or 832) comprises a conductive material suitable for use as aworking electrode or, preferably, a counter electrode in an ECL assay.Plate top 802 (or 848) may be made of a conducting material such asmetal or a carbon-containing plastic composite. Alternatively,conductivity is achieved via a conductive coating such as a metal film(e.g., an evaporated, electrodeposited or electroless deposited metalfilm) or a metal-containing paint such as a silver paint. Sectoring ofthe plates into individually addressed sectors of jointly addressablewells can be achieved by sectioning conductive layer 820 (or 858) and/orplate top 802 (or 832) into electrically isolated sections. The platetop may be sectioned by division into individual pieces or by patterningof a conductive coating on a contiguous piece.

In operation, test samples are introduced into wells of plate 800 (or830). A source of electrical energy is connected to plate top 802 (or832) and one or more sections of conductive layer 820 (or 858).Preferably, the connection to plate top 802 (or 832) is made bycontacting the side of the plate. Preferably, the connections to the oneor more sections of conductive layer 820 (or 858) are made by contactingthe bottom of the plate. Application of electrical energy across theseconnections leads to the application of an electrochemical potentialacross the test samples via the exposed surfaces of plate top 802 (or832) and conductive layer 820 (or 858) (the application ofelectrochemical potential being confined to wells in sectors contactingthe one or more sections of conductive layer 820 or 858). In the case ofan ECL assay, it is preferable to apply electrical energy so as togenerate ECL at or near the surface of conductive layer 820 or 858(i.e., conductive layer 820 or 858 provides the working electrode) sothat light is generated near the center of the well.

5.2.5 Multi-Layer Electrode Plates Having Conductive Through-Holes

FIG. 9A shows an exploded view of multi-well assay plate 930, anembodiment of multi-well assay plate 500 from FIG. 5 that comprisesadditional adaptations allowing for convenient electrical connections tothe conductive layers of the plate. FIG. 9B shows a stylizedcross-sectional view of three wells 942A, 942B and 942C of the sameplate. Plate 930 comprises a laminar structure comprising, in sequence,plate top 932, adhesive layer 944, second conductive layer 948,dielectric layer 950, first conductive layer 958, substrate layer 959and contact layer 960. Holes 934, 946, 949 and 956 through plate top932, adhesive layer 944, second conductive layer 948 and dielectriclayer 950, respectively, are aligned so as to form a plurality of wellshaving well bottoms defined by first conductive layer 958. Conductivethrough-holes 962 through substrate 959 provide a conductive pathbetween first conductive layer 958 and the portion of contact layer 960that define electrical contacts 963 (963A being, preferably, workingcontacts and 963B being, preferably, counter contacts). Conductivethrough-holes 964 and 966 through substrate 959 and dielectric layer950, respectively, provide a conductive path between second conductivelayer 948 and the portion of contact layer 960 that define electricalcontacts 965. Electrical connection to conductive layers 948 and 958can, therefore be made from the bottom of the plate by contactingelectrical contacts 965 and 963, respectively. Element 970 (FIG. 9A)shows layers 960, 959, 958, 950 and 948 aligned and stacked, in orderfrom top to bottom—948 (top), 950, 958, 959, and 960 (bottom)—so as toform a plate bottom with integrated electrodes.

Plate top 932 is a plate top analogous to plate top 502 in FIG. 5.Adhesive layer 944 is an adhesive suitable for forming a fluid-tightseal between plate top 932 and second conductive layer 948. Adhesivelayer 944 may be an adhesive coating applied, e.g., by spray coating,onto plate top 932 or second conductive layer 948. In a preferredembodiment, adhesive layer 944 is a double sided adhesive tape (i.e., aplastic film coated on both sides with adhesive). Holes 946 arepreferably formed by a cutting process such as laser drilling or diecutting. Optionally, adhesive 944 may be omitted (when second conductivelayer 948 or plate top 932 have adhesive properties or when sealing isaccomplished without the use of adhesives, e.g., by clamping, heatsealing, sonic welding, solvent welding, etc.). Alternatively, bothplate top 932 and adhesive 944 may be omitted. Second conductive layer948 is a material suitable for use as a working electrode, or preferablya counter electrode in an ECL assay (see for example the description ofthe analogous second conductive layer 504 in FIG. 5). Preferably, it isa conductive coating such as a carbon ink and may be formed by aprinting process such as ink jet printing, laser printing, or, mostpreferably, screen printing. Second conductive layer 948 is preferablyof sufficient conductivity so that, during use of the plate in an assay,an electrical potential applied to second conductive layer 948 will beevenly distributed over its surface. To attain suitably highconductivity with a moderately conductive electrode material such as acarbon ink, it may be advantageous that second conductive layer 948comprise two layers: i) a highly conductive underlayer such as a silverpaint and ii) an electrode material overlayer such as carbon ink. Whenforming such layers, e.g., by a two step printing process, it isbeneficial that the overlayer be thick enough and of suitable dimensionsto ensure that a sample in wells 932 is not exposed to the underlayermaterial. Dielectric layer 956 is an electrically insulating filmsuitable for preventing electrical contact between conductive layers 948and 958. Preferably, dielectric layer 956 is comprises dielectric inkand is formed by a printing process such as screen printing and,optionally, UV curing.

First conductive layer 958 is a material suitable for use as a counterelectrode, or preferably a working electrode in an ECL assay (see forexample the description of the analogous first conductive layers 508 inFIGS. 5 and 700, 720, 740 and 760 in FIGS. 7A-7D). Preferably, firstconductive layer 958 comprises a conductive coating such as a carbon inkand may optionally comprise a highly conductive underlayer, such assilver ink, to better distribute electrical potential across the layerduring the course of an assay. Such one or two layer coatings may beformed by printing processes such as screen printing and are preferablydesigned so as to ensure that samples in wells 932 do not contact theunderlayer. First conductive layer 958 is sectioned into 12 electricallyisolated columnar sections corresponding to a column of wells in plate930; such sectioning may be achieved via patterned printing, e.g., byscreen printing. Substrate 959 is a non-conducting material such as anon-conducting plastic sheet. Through-holes 962 and 964 in substrate 959are, preferably, made by a cutting process such as die cutting or laserdrilling. Through-holes 962 are filled with a conductive material toprovide an electrical connection between electrical contacts 963 andfirst conductive layer 958. Through-holes 964 and 966 are filled withconductive material to provide an electrical connection between contacts965 and second conductive layer 948 (holes 964 and 966 are located inthe regions between the sections of first conductive layer 958 so as toensure that that they are electrically isolated from first conductivelayer 958). Through-holes 962, 964 and 966 are preferably filled withconductive material during the formation of conductive layers 960, 958and/or 948, e.g., during the screen printing of a conductive ink on asubstrate, excess ink is forced into holes in the substrate so as tofill the holes with the conductive ink. Contact layer 965 is aconductive material such as a conductive ink. Preferably it is a screenprinted silver paint with a screen printed carbon ink overlayer toprevent corrosion of the silver. We have also found that the presence ofexposed silver appears to negatively influence the plasma treatment ofsurfaces (even on the opposite side of the plate); therefore, whenplasma treatment is used to modify a surface of the assay plate it isparticularly advantageous that there be no exposed silver.

In operation, test samples are introduced into wells of plate 930. Asource of electrical energy is connected to second conducting layer 948and one or more sections of conductive layer 958 (via electricalcontacts 965 and one or more of electrical contacts 963, respectively).Application of electrical energy across these connections leads to theapplication of an electrochemical potential across the test samples viathe exposed surfaces of conducting layers 948 and 958 (the applicationof electrochemical potential being confined to wells in sectorscontacting the one or more sections of conductive layer 958). In thecase of an ECL assay, it is preferable to apply electrical energy so asto generate ECL at or near the surface of conductive layer 958 (i.e.,conductive layer 958 provides the working electrode) so that light isgenerated near the center of the well.

5.2.6 Single-Layer Electrode Plates Having Conductive Through-HOLES

Another aspect of the invention relates to multi-well plates having oneor more printed electrodes. Thus, one embodiment of the inventionrelates to a multi-well plate comprising:

-   -   (a) a substrate surface;    -   (b) one or more working electrodes on the substrate surface;    -   (c) one or more counter electrodes on the substrate surface; and    -   (d) a plate top having plate top openings;

wherein the plate top openings are positioned on the substrate surfaceso as to form a plurality of wells having at least one working electrodeand at least one counter electrode.

Preferably, the electrodes are printed, most preferably screen printed,onto substrate. Preferably, the electrodes comprise carbon ink.

Another embodiment relates to a multi-well plate having a plurality ofwells comprising a first electrode surface formed by applying two ormore conductive layers comprising carbon.

Preferably, three or more layers of carbon are formed. Preferably, apatterned working electrode surface is formed by applying a first layerof carbon and a second layer of carbon, wherein the area of one layer ofcarbon is greater than the area of the other layer of carbon.

Preferably, the wells comprise a working electrode surface formed byapplying one or more layers of carbon onto a conductive layer comprisingsilver. Preferably, the one or more layers of carbon completely coverthe conductive layer.

FIGS. 10A and 10B show another embodiment of the multi-well assay plateof the invention. Multi-well assay plate 1000, is similar to multi-wellassay plate 1600 from FIG. 16A but comprises additional adaptationsallowing for convenient electrical connections to the conductive layersof the plate. Multi-well assay plate 1000 is a laminar structurecomprising, in sequence, a plate top 1020, an adhesive layer 1030, adielectric layer 1040, a conductive layer 1050, a substrate layer 1060and a contact layer 1070. Holes 1022 and 1032 through plate top 1020 andadhesive layer 1030, respectively, are aligned so as to form a pluralityof wells 1002 having well bottoms defined by dielectric layer 1040,conductive layer 1050 and/or substrate layer 1060 and well walls definedby the interior surfaces of holes 1022 and 1032. Through-holes 1062 and1064 through substrate layer 1060 provide an electrical path betweenelements of conductive layer 1050 and elements of contact layer 1070.Element 1080 shows layers 1070, 1060, 1050 and 1040 aligned and stacked,in order from top to bottom—1040 (top), 1050, 1060, and 1070 (bottom)—soas to form a plate bottom with integrated electrodes.

Plate top 1020 is a plate top analogous to plate top 502 in FIG. 5.Adhesive layer 1030 is an adhesive suitable for forming a fluid-tightseal between plate top 1020 and dielectric layer 1040, conductive layer1050 and/or substrate layer 1060. Adhesive layer 1030 may be an adhesivecoating applied, e.g., by spray coating, onto plate top 1020. In apreferred embodiment, adhesive layer 1030 is a double sided adhesivetape (i.e., a plastic film coated on both sides with adhesive). Holes1032 are preferably formed by a cutting process such as laser drillingor die cutting. Optionally, adhesive 1030 may be omitted (e.g., when theadjoining layers have adhesive properties or when sealing isaccomplished without the use of adhesives, e.g., by clamping, heatsealing, sonic welding, solvent welding, etc.). Alternatively, bothplate top 1020 and adhesive layer 1030 may be omitted.

Conductive layer 1050 comprises materials suitable for use as workingelectrodes and/or counter electrodes in an ECL assay and is supported onsubstrate 1060, a non-conductive substrate such as a plastic sheet orfilm. Preferably, conductive layer 1050 is a conductive coating such asa carbon ink and may be formed by a printing process such as screenprinting. Conductive layer 1050 is sectioned, e.g., by screen printingin a defined pattern, into 12 electrically isolated working electrodesections 1052 and 13 electrically connected counter electrode sections1054. As shown in the figure, the sectioning is designed so that fluidin a given well will be in contact with at least one working electrodesection and at least one counter electrode section. The workingelectrode sections may have a different composition than the counterelectrode sections so as to optimize the performance of the electrodesor they may comprise the same materials so as to minimize the complexityof manufacturing, e.g., to reduce the number of printing steps.Preferably, they both comprise a carbon ink overlayer over a silver inkunderlayer; the carbon ink providing the active electrode surface andthe silver ink providing sufficient conductivity so that, during use ofthe plate in an assay, electrical potential is evenly distributedthroughout a particular section. When forming such layers, e.g., by atwo step printing process, it is beneficial that the overlayer be ofslightly larger dimensions than the underlayer and that it be ofsuitable thickness to ensure that a sample in wells 1002 is not exposedto the underlayer material. It may be beneficial to print or deposit theoverlayer in multiple layers so as to ensure that the underlayer iscompletely covered so that the underlayer does not interfere withsubsequent processing steps or with ECL measurements (e.g., a preferredelectrode material comprises three layers of carbon ink over a layer ofsilver ink, the layers most preferably being deposited by screenprinting). Dielectric layer 1040 is an electrically insulating film,preferably formed from a dielectric ink by a printing process such asscreen printing. Dielectric layer 1040 is patterned so as to define thesurfaces of conductive layer 1050 that contact fluids in wells 1002(i.e., the surfaces that are not covered). Holes 1042 in dielectriclayer 1040 define fluid containment regions on the working electrodesections 1052 of conductive layer 1050. In such fluid containmentregions, the dielectric layer acts as a barrier that can be used toconfine small volumes of fluids over the working electrode. Optionally,dielectric layer 1040 may be omitted.

Contact layer 1070 is a conductive layer that allows for electricalconnection of the multi-well assay plate to an external source ofelectrical energy. The contact layer is sectioned in a series of workingelectrode contacts 1072 and counter electrode contacts 1074 to allowindependent connection to specific sections of electrodes 1052 and 1054.The contact layers are, preferably, formed by printing, most preferablyscreen printing, a silver ink under layer (to provide high conductivity)followed by a carbon ink overlayer (to prevent corrosion of the silverink and prevent any deleterious effects by the exposed silver on asubsequent plasma processing step). Holes 1062 and 1064 in substrate1060 are, preferably, made by a cutting process such as die cutting orlaser drilling. Holes 1062 are filled with a conductive material toprovide an electrical connection between working electrode contacts 1072and working electrode sections 1052. Holes 1064 are filled withconductive material to provide an electrical connection between counterelectrode contacts 1074 and counter electrode sections 1054. Holes 1062and 1064 are preferably filled with conductive material during theformation of conductive layer 1050 or contact layer 1070, e.g., duringthe printing of a conductive ink on a substrate, excess ink is forcedinto holes in the substrate so as to fill the holes with the conductiveink.

In operation, test samples are introduced into wells of plate 1000. Asource of electrical energy is connected across one or more workingelectrode sections 1052 and one or more counter electrode sections 1054(via one or more of working electrode contacts 1072 and one or more ofcounter electrode contacts 1074, respectively). Application ofelectrical energy across these connections leads to the application ofan electrochemical potential across the test samples via the exposedsurfaces of electrode sections 1052 and 1054 (the application ofelectrochemical potential being confined to wells in sectors contactingworking electrode and counter electrode sections that are in electricalconnection to the source of electrical energy).

Plate 1000 as shown in FIGS. 10A and 10B is a 96-well plate divided into12 independently addressable sectors of 8 wells (i.e. 12 columns of 8wells). The structure shown in FIGS. 10A and 10B is readily modified soas to be applicable to plates having different numbers of wells,different arrangements of wells and/or different arrangements ofindependently addressable sectors.

FIGS. 11A and 12A show two alternative embodiments. FIG. 11A shows amulti-well assay plate 1100 that is analogous in structure and functionto plate 1000 except that the components are configured so as to dividethe plate into six independently addressable square sectors each havinga 4×4 array of wells. Element 1180 shows layers 1140, 1150, 1160 and1170 aligned and stacked, in order from top to bottom, 1140 (top), 1150,1160 and 1170 (bottom). FIG. 12A shows a multi-well assay plate 1200that is analogous in structure and function to plate 1100 except thatthe components are configured so as to provide 384 wells in a 24×116array. Element 1280 shows layers 1240, 1250, 1260 and 1270 aligned andstacked, in order from top to bottom, 1240 (top), 1250, 1260 and 1270(bottom).

The electrode patterns illustrated in details C of FIGS. 11A and 12A(i.e., the patterns used for conductive layers 1150 and 1250,respectively) illustrate some useful concepts in the design of suitableelectrode patterns. In general, the electrode material should be ofsufficient conductivity that potential drops along the surface of theelectrodes are small relative to the potential drops between opposingelectrodes. By proper electrode design it is possible, although notrequired, to make additional compensations for these small potentialdrops along the surface of the electrode. FIG. 12A shows the division ofconductive layer 1250 into i) electrodes 1254 that, preferably, providethe counter electrode surface within wells of the plate and ii)electrodes 1252 that, preferably, provide the working electrodes withinwells of the plate. The electrodes are divided into electrode stripsthat run the length of a given plate sector. Each full-width strip ofelectrode 1252 is matched with two half-width strips of electrode 1254so that the overall electrical resistance along the length of the sectoris evenly matched between the opposing electrodes. The opposingelectrodes are contacted at opposite ends of the sector so the overallresistance in the leads to any particular well should be a constantvalue. Any potential drop due to this resistance should therefore beconstant and should not cause variability between wells. Similarly, theresistance in electrodes 1152 and 1154 as shown in FIG. 11A are matchedalong the length of the sector, in this case the resistance matching isaccomplished by patterning electrodes 1154 so that the length of theelectrodes comprises wide regions (to maximize electrode surface areawithin the wells) alternating with narrow regions (to help match theoverall resistance of along the length of electrodes 1154 with that ofelectrodes 1152).

5.2.7 Specific Embodiments of Plates Having Wells Divided into aPlurality of Assay Domains

In some embodiments of the invention, the active area of the workingelectrode in a well of a multi-well assay plate is divided into aplurality of assay domains. For example, a working electrode used in anECL binding assay may have immobilized on distinct regions of itssurface a plurality of different binding reagents so as to form aplurality of distinct binding domains differing in their affinity foranalytes of interest. Wells having such electrodes allow a number ofdifferent analytes to be measured concurrently in the same sample in thesame well (e.g., by imaging the light emitted from the well andcorrelating the amount of each analyte of interest to the light emittedfrom an assay domain specific for that analyte). A patterned dielectricmay be used to facilitate the division of the working electrode area ina well into one or more assay domains; the assay domains are defined byone or more holes in a dielectric layer covering the working electrode.The dielectric layer providing a barrier that can confine small volumesof fluid to the assay domains formed by the regions of exposed workingelectrode (also referred herein as fluid containment regions). The useof dielectric layers to form such assay domains is described in moredetail in the description of FIGS. 4A-4E. Microdispensing of fluids ontoselected fluid containment regions allows for the selectiveimmobilization of reagents in specific fluid containment regions or theconfinement of certain steps of an assay to specific fluid containmentregions.

FIGS. 13A-13B, 14A-14B and 15 show examples of multi-well assay platesof the invention that have a plurality of fluid containment regions ineach well. FIG. 13A shows multi-well assay plate 1300, a plate analogousto multi-well assay plate 500 (as shown in FIG. 5) except that thepattern of holes 1312 through dielectric layer 1306 has been modified todefine a plurality of fluid containment regions over the workingelectrode surface (i.e., first conductive layer 1308). FIG. 13B showsmulti-well assay plate 1350, a plate analogous to multi-well assay plate500 (as shown in FIG. 5) except that the pattern of holes 1360 in secondconductive layer 1354 and the pattern of holes 1362 in dielectric layer1356 has been modified to define a plurality of fluid containmentregions over the working electrode surface (i.e., first conductive layer1358). The modifications of FIGS. 13A and 13B can also by analogy beintroduced into the specific embodiments of the invention described byFIGS. 8A-8C, 9A-9B, 10A-10B, 11A and 12A. FIGS. 14A-14B shows amulti-well assay plate 1400, a plate analogous to multi-well assay plate1600 (as shown in FIGS. 16A-16C) except that the pattern of holesthrough dielectric layer 1406 has been modified to define a plurality offluid containment regions 1407 over the working electrode surface (i.e.,working electrode section 1422). Element 1412 shows layers 1406, 1408and 1410 aligned and stacked, in order from top to bottom, 1406 (top),1408 and 1410 (bottom). The modification of FIGS. 14A-14B can also byanalogy be introduced into the specific embodiments of the inventiondescribed by FIGS. 10A-10B; 11A-12A.

FIGS. 14A-14B does not show sectoring or conductive contacts, however,such sectoring and/or conductive contacts may be introduced as describedabove, e.g., by analogy to FIGS. 10A-10B and/or 11A. FIG. 15 shows,multi-well assay plate 1500, an embodiment of the invention that isparticularly well suited for genomic or proteomic analysis. Multi-wellassay plate 1500 is an adaptation of plate 1300 having only sixindependently addressable square wells. The size of the wells is chosenso as to optimize the efficiency of the imaging of luminescencegenerated from the wells by the imaging instrument (as described below).Multi-well assay plate 1500 is a laminar structure comprising, insequence, plate top 1520, adhesive layer 1530, conductive tape layer1514B, dielectric layer 1540, conductive layer 1552, substrate 1560,contact layer 1572 and conductive tape layer 1514A. Element 1580 showslayers 1572, 1560, 1552 and 1540 aligned and stacked, in order from topto bottom, 1540 (top), 1552, 1560,1572 (bottom). Conductive tape layers1514A and 1514B are provided by folding conductive tape 1510 aroundelement 1580 at fold 1516 (by analogy to FIG. 8A). Holes 1522, 1532 and1518 are aligned so as to form a plurality of wells having well bottomsdefined by element 1580. Through-holes 1562 through substrate 1560provide an electrical path between conductive layer 1552 and contactlayer 1572. Through holes 1512 through conductive tape layer 1514Aprovide access to contact layer 1572 (and, therefore a way to contactconductive layer 1552). Plate top 1520 is analogous to plate top 1020from FIGS. 10A-10B except for the specific arrangement of wells.Adhesive layer 1530 is an adhesive analogous to adhesive layer 1030 inFIGS. 10A-10B and may be omitted. Conductive tape 1510 is analogous toconductive tape 810 as described in FIG. 8A. Substrate 1560, conductivelayer 1552, dielectric layer 1540 and contact layer 1572 are similar incomposition and preparation to substrate 1060, conductive layer 1050,dielectric layer 1040 and contact layer 1072 as described for FIG. 10.Conductive layer 1552 is sectioned into 6 square sections so as todivide plate 1500 into 6 independently addressable sectors (each havingone well). Holes 1542 through dielectric layer 1540, define a largenumber (preferably 10-50,000, more preferably 100-10,000; 256 are shownin the figure) of fluid containment regions in each well. Bindingreagents such as specific nucleic acid sequences or specific proteinscan be selectively introduced and or immobilized into specific fluidcontainment regions by selectively microdispensing the binding reagentsinto the specific fluid containment regions.

While the figures illustrating embodiments of the plates of theinvention have shown specific patterns for number, shape anddistribution of wells, sectors and fluid containment regions/assaydomains, it should be clear that the designs are adaptable so as toallow for a wide variation in these parameters.

5.3 Apparatus for Reading Multi-Well Assay Plates

Another aspect of the invention relates to an apparatus for measuringluminescence, preferably electrode induced luminescence, more preferablyelectrochemiluminescence, from a multi-well assay plate having aplurality of wells or a single-well plate having a plurality of assaydomains within a single well. Although the apparatus is configured forelectrode induced luminescence, such an apparatus can also beadditionally configured to other luminescence assays such aschemiluminescence and/or fluorescence assays.

Preferably, the apparatus is adapted to measure light from at least aportion of the plurality of wells. Preferably, the portion comprises oneor more, more preferably two or more, even more preferably four or moreand most preferred eight or more, and, preferably, less then all, of thewells of the plate.

The apparatus may comprise a source of electrical energy for generatingluminescence within at least a portion of the plurality of wells,preferably the source of electrical energy is applied as an electricalvoltage or current to the portion of the plurality of wells. Preferably,the portion comprises one or more, more preferably two or more, evenmore preferably four or more and most preferred eight or more, andpreferably less then all, of the wells of the plate. The source ofelectrical energy may include a power cable, power source, powergenerator, battery or other energy storage media or the like.Preferably, the source of electrical energy includes an electricalsubsystem (for example, a current source, voltage source, or currentand/or voltage waveform generator) capable of providing current and/orvoltage to one or more of the plurality of wells. It is understood thatthe term “source of electrical energy” includes devices and apparatuseswhich require that such source be connected to an external power supply(e.g., a wall socket). According to one preferred embodiment, the sourceof electrical energy is capable of delivering a potential to the workingelectrode relative to the counter electrode of from 0 to +8 Volts DC.Preferably, the source of electrical energy has a voltage resolutionless than or equal to 50 mV, preferably 20 mV, even more preferably 10mV and most preferred 5 mV. According to another preferred embodiment,the source of electrical energy is capable of generating voltagewaveforms consisting of ramps (constant dV/dt).

The apparatus may also include a support or plate holder adapted to holdthe multi-well assay plate. Preferably, the support comprises a carrier(e.g., a drawer) adapted to carry the plate into and/or through theapparatus, preferably into and/or through a light tight enclosure withinthe apparatus.

According to another embodiment, the apparatus further comprises amotion control subsystem (preferably comprising one or more computers,linear actuators or translation tables having one, two, three or moreaxis of motion, and/or motors for driving the motion) for moving platesin and out of the apparatus and for correctly aligning the plate withlight detectors and/or electrical contacts within the apparatus.Preferably, the motion control subsystem provides independent control ofat least four stepper motors. Preferably, the motion control subsystemis capable of independently controlling the maximum plate carriervelocity. Moreover, the motion control subsystem preferably allows forcontrolled acceleration for each motion axis and/or provides integrationof a plate position encoder on each motion axis and/or is adapted toallow the plate position of each axis to be verified using a positionencoder. Preferably, the motion control subsystem provides for stalldetection using a position encoder. The plate motion control subsystempreferably (i) has a movement resolution less than or equal to 0.01inches, preferably less than or equal to 0.005 inches, even morepreferably less than or equal to 0.001 inches, (ii) is capable ofproviding continuous motion of at least 1 inch per second, preferably atleast 5 inches per second and/or (iii) is capable of placing the platewithin a circular tolerance zone within within 0.01 inches, morepreferably within 0.005 inches and even more preferably within 0.001inches.

In one embodiment of the invention, the apparatus includes one or moreelectrical connectors (optionally included as part of a plate contactsubsystem) adapted to connect the source of electrical energy to thewells and/or plate support. Preferably, the connectors are adapted tocontact the bottom of the multi-well plate. Preferably, the apparatuscomprises two or more electrical connectors, more preferably between 3and 20 electrical connectors, even more preferably comprises sixelectrical connectors. The electrical connectors may be incorporatedwithin a plate holder so that electrical connection to the plate isachieved by placing the plate in the plate holder or the electricalconnectors may be in separate components. Alternately, some electricalconnectors may be incorporated within the plate holder and some may beincluded in separate components.

According to one preferred embodiment, the apparatus comprises a 2×3array of electrical connectors, preferably the 2×3 array comprises fourworking electrical connectors and two counter electrical connectors.According to another preferred embodiment, the apparatus comprises sevenelectrical connectors, preferably a linear array of seven electricalconnectors, most preferably a linear array of four working electricalconnectors and three counter electrical connectors. In anotherembodiment, one or more electrical connectors (preferably two or more,more preferably between 3 and 20) are incorporated within a plate holderso that electrical connection to the plate is achieved by placing theplate in the plate holder.

Preferably, the electrical connectors and/or plate contact subsystem areadapted to make contact to the bottom of an assay plate. Optionally, atleast one electrical contact is made to the top or side of the plate.Preferably the upward movement of a plate due to force of the electricalcontacts and/or plate contact subsystem is less than 0.010 inches.According to another embodiment, the plate contact subsystem has aminimum step resolution of at least 0.004 inches.

The emitted luminescence is preferably measured to determine, forexample, the presence or absence or amount of analyte of interest in oneor more samples. The apparatus may comprise a light detector formeasuring the luminescence within at least a portion of the plurality ofwells. Alternatively, the apparatus may comprise a structure (e.g., aslot or the like) for inserting or adjoining a suitable light detectorsuch as film. Such an apparatus would include all the other componentsof the system, but the user would add the light detector. Preferably,such an apparatus would be adapted to be suitably mated with one or morelight detectors.

One preferred embodiment of the invention incorporates a light detectorfor measuring emitted luminescence from at least a portion of theplurality of wells. Preferably, the apparatus includes both a lightdetector and a source of electrical energy for generating luminescence,more preferably electrochemiluminescence, within the plurality of wellsand a light detector for measuring emitted luminescence. Advantageously,such an apparatus may also include the electrical connectors adapted toconnect the source of electrical energy to the wells.

One aspect of the invention relates to apparatus capable of measuringluminescence and/or generating luminescence in sectors. For example, theapparatus may induce luminescence and/or measure emitted luminescence inless than the entire plate and/or less than all of the wells on theplate. The term “sector” as used herein is defined as independentlyaddressable sectors of jointly addressable wells. Preferably, a “sector”comprises one or more wells, two or more wells, less than all the wells,and/or less than 50% of the wells.

Thus, one embodiment of the invention relates to an apparatus formeasuring luminescence from a multi-well assay plate having a pluralityof independently addressable sectors of jointly addressable wells.

According to one embodiment, the apparatus includes one or moreelectrical connectors adapted to connect the source of electrical energyto the independently addressable sectors. Preferably, the apparatus alsoincludes a plate holder for holding the plate and the electricalconnectors and the plate holder are adapted to move relative to oneanother to allow for sequentially contacting the sectors. In analternate embodiment, one or more electrical connectors (preferablybetween 3 and 20) are incorporated within the plate holder so thatelectrical connection to one or more sectors of the plate is achieved byplacing the plate in the plate holder.

According to one embodiment, the apparatus comprises a plate holderadapted to hold the plate onto a measuring platform or a detectionlocation (e.g., where the luminescence is induced and/or detected)during the detection step and a plurality of electrical connectorsadapted to contact the plate, thereby providing electrical energy to thewells. Preferably, the electrical connectors contact the bottom surfaceof the plate. Advantageously, the contacting occurs between the wells,preferably by pushing against the well walls, i.e., where the plate ismost rigid.

The apparatus may include a plate holder adapted to hold the plate ontoa measuring platform during the measuring. This is advantageous when theelectrical connectors contact the plate bottom since the holder may beconfigured to hold the plate down and/or to prevent the contacts fromlifting the plate. This is important, for example, when an imagingsystem is employed to image the luminescence from the wells. If theconnectors were allowed to lift or otherwise move the plate, the imagemay be distorted.

According to another embodiment, the apparatus comprises a lightdetector and a support adapted to hold the multi-well assay plate,wherein the light detector and the support are adapted to move relativeto one another to allow for sequentially measuring the sectors.

Another aspect of the invention involves the use of an imaging system toimage emitted luminescence. Preferably, the apparatus further comprisesa computer image analyzer. According to one preferred embodiment, thecomputer has software for subtracting background light and/oreliminating cosmic ray induced artifacts and/or any defects in thephotodetector.

One embodiment of the invention relates to an apparatus for measuringluminescence from a multi-well assay plate having a plurality of wallscomprising:

-   -   (a) an imaging system comprising a camera, the imaging system        adapted to image at least a portion of the plurality of wells        and thereby measure the luminescence; and    -   (b) a source of energy for generating luminescence within at        least a portion of the plurality of wells, the source of        electrical energy applied as an electrical voltage or current to        the portion of the plurality of wells.

Another relates to an apparatus for measuring luminescence from amulti-well assay plate having a plurality of wells comprising:

-   -   (a) an imaging system comprising a camera, the imaging system        adapted to image at least a portion of the plurality of wells        and thereby measure the luminescence; and    -   (b) a source of electrical energy adapted to provide electrical        energy to the plurality of wells in sectors.

Another embodiment of the invention relates to an apparatus formeasuring luminescence from a multi-well assay plate having a pluralityof wells comprising:

-   -   (a) an imaging system comprising a camera, the imaging system        adapted to image the plurality of wells in sectors and thereby        measure the luminescence in sectors; and    -   (b) a source of electrical energy, the source of electrical        energy applied as an electrical voltage or current to the        portion of the plurality of wells.

Accordingly, the invention includes an apparatus for measuringluminescence from a multi-well assay plate having a plurality of wellscomprising an imaging system comprising a camera, the imaging systemadapted to image at least a portion of the plurality of wells of themulti-well assay plate. Preferably, the apparatus further comprises asupport adapted to hold the multi-well assay plate in a detectionposition where the camera can image the portion. Advantageously, thecamera and/or the support are adapted to image the plurality of wells insectors and thereby measure the luminescence. Preferably, the apparatusfurther comprises a camera mounting system for positioning the cameraand/or any associated optics (e.g., lenses). Preferably, the cameramounting system maintains the camera imaging surface of an imagingsystem and/or the associated optics perpendicular to the multi-wellplate within plus or minus 5 degrees, more preferably within plus orminus 3 degrees, even more preferably within plus or minus 2 degrees andmost preferably within plus or minus 1 degree.

Providing an apparatus, method and plate, which enables the luminescenceto be measured in sectors, allows for greater light collectionefficiency. Accordingly, one preferred embodiment of the inventionrelates to an apparatus comprising an imaging system adapted tosimultaneously image emitted luminescence from at least two of theplurality of wells, wherein the imaging collects a cone of luminescencehaving a cone full angle of at least 10 degrees, preferably at least 15,more preferably at least 20, even more preferably at least 25, and mostpreferred at least 30 degrees.

According to another embodiment, the apparatus further comprises asupport adapted to hold the multi-well assay plate in a detectionposition and/or electrical connectors adapted to connect the multi-wellassay plate to the source. Preferably, the apparatus is adapted toconnect the electrical connectors to a plurality of sectors and/or imagethe plurality of sectors sequentially.

Another aspect of the invention relates to an apparatus or method, whichemploy an array of light detectors, preferably an array of discretelight detectors such as an array of photodiodes.

Thus, one embodiment of the invention relates to an apparatus formeasuring luminescence from a multi-well assay plate having a pluralityof wells comprising:

(a) an array of light detectors adapted to detect light from at least aportion of the multi-well assay plate, preferably in sectors; and/or

(b) a source of electrical energy for providing electrical energy to themulti-well plate, preferably in sectors;

wherein the apparatus preferably induces and/or measures theluminescence in sectors.

Another embodiment relates to an apparatus further comprising:

-   -   (a) a support adapted to hold the multi-well assay plate in a        detection position; and/or    -   (b) electrical connectors adapted to connect the sector of the        multi-well plate to the source;        -   wherein the apparatus is preferably adapted to connect the            electrical connectors to the plurality of sectors and/or            detect luminescence from the plurality of sectors            sequentially.

Preferably, wherein the apparatus is adapted to allow the array of lightdetectors to move relative the support so as to allow for alignment ofeach sector with the array of detectors.

According to a preferred embodiment, the apparatus comprises onedetector per well per sector. Preferably, the array of light detectorsis adapted to be aligned with an array of wells. For example, referringto FIG. 1, a linear array of eight appropriately sized photodiodes couldbe aligned with a row of wells. Preferably the light detector array is alinear array, which can be linearly scanned across the plate.

According to another embodiment, the apparatus is adapted to use modules(preferably plates) where the working electrode and/or the counterelectrode on the module is replaced with one or more probes provided bythe apparatus, preferably an array of working electrode probes and/orcounter electrode probes, which are inserted into the wells to provideelectrical energy to the wells. A single probe could be aligned andarranged so as to provide electrical energy to one well of a multi-wellplate at a time or an array of probes could be used to provideelectrical energy to a plurality of wells (e.g., the array could be usedto provide electrical energy to one group of wells and then be moved toprovide electrical energy to a different group of wells). Preferably,the probes comprise one or more fiber optic probes coated in anelectrode material so as to function both as electrodes (preferably acounter electrode) and conduits for conveying light generated in wellsto one or more light detectors in the apparatus.

According to one preferred embodiment, the apparatus further comprisesone or more robotic and/or computer systems adapted to perform one ormore of the following functions: (i) moving assay modules; (ii) shakingthe assay modules (and assay contents therein); (iii) storing plates(e.g., refrigeration unit); (iv) liquid or reagent handling (e.g.,mixing reagents); and (v) reagent delivery (e.g., dispensing reagentsinto wells, etc.).

FIG. 17 illustrates an embodiment of the apparatus of the presentinvention. Reader 1700 comprises a cover (or case) 1702, a light tightenclosure 1704 with one or more doors and/or apertures 1714, aphotodetector 1706, optics 1708, multi-well assay plate 1710, platealignment mechanism 1712, plate transport mechanism 1716, bar codereader 1718, electronics 1720, current/voltage source 1722, plateelectrical connector 1724, computer 1726, power supply 1728, data andnetwork connections 1730, indicators 1732, reagent handler 1734, one ormore plate stackers 1736, robotics 1738, and plate carrier 1740.Preferably, the majority of cover 1702 is a molded structure made fromrigid plastic materials such as polyurethanes, structural foams, ABS,polystyrenes, polypropylene, polycarbonates and the like. Cover 1702 mayalso incorporate metals (e.g., aluminum, brass, steel), composites (e.g.carbon fiber composites, polymer composites), and/or carbon basedmaterials. Cover 1702 may also be painted; conductive paints (e.g.,paints containing metal flake) may be used to reduce electromagneticinterference (i.e., as EMI shielding). The cover, preferably, functionsto enclose, support and protect certain elements of the reader. Thecover may incorporate vents or other openings and may also include oneor more fans for cooling the instrument and/or for maintaining thecirculation of air through the instrument. In a preferred embodiment thecover provides separate intake and exhaust vents for coolingphotodetector 1706.

Light tight enclosure 1704 is a sealed compartment designed to preventthe entrance or exit of light. Preferably, the majority of light tightenclosure 1704 is comprised of a rigid material such as steel oraluminum. In a preferred embodiment, light tight enclosure 1704 iscomprised of aluminum sheet metal. Light tight enclosure 1704 may alsoincorporate non-rigid or compliant materials. In a preferred embodiment,light tight enclosure 1704 contains a compliant closed cell foam gasketthat acts as a seal to prevent passage of light. Light tight enclosure1704 has one or more doors and/or apertures 1714 and through whichmulti-well assay plates of the invention may pass during operation ofthe reader. Aperture 1714 incorporates a door that opens to allowtransport of multi-well assay plates into and out of the reader. Thedoor opens and closes by sliding along a tongue and groove configurationat the junction between the door and aperture 1714. The tongue andgroove configuration provides a tortuous path that reduces transmissionof light. The movement of the door or aperture 1714 is mechanicallydriven by a linear actuator that is controlled by computer 1726 andelectronics 1720. Light tight enclosure 1704 is joined to optics 1708,or if optics 1708 are omitted, to photodetector 1706. Enclosure 1704provides a compliant coupling between optics 1708 (or photodetector1706) that allows focusing of the emitted light onto the photodetector(e.g., by focusing a lens) without disrupting the light tight enclosure.This compliant coupling may include one or more baffles, light tightseals or light tight flexible housings. In a preferred embodiment, theflexible coupling is a slipping light tight seal comprised of a siliconegasket or layer. According to another preferred embodiment, the couplingcomprises flexible, light-tight bellows (preferably made of neoprene) atthe lens-light tight enclosure interface. The bellows allows easierfocusing and motion of the lens while still providing a light tightseal. Light tight enclosure 1704 can be dismantled without disturbingthe optics 1708 and/or photodetector 1706. The walls of the light tightenclosure are preferably black to reduce reflection of light.Preferably, the light tight enclosure is adapted to provide at least adegree of external light rejection so that a change in ambient lightlevel from 500 lux to 0 lux does not increase the apparent coefficientof variation in background signal by more than 20%, more preferably bymore than 15%, even more preferably by more than 10% and most preferredby more than 5%.

Photodetector 1706 primarily measures the light emitted from multi-wellassay plates during the conduct of electrochemiluminescent assays inreader 1700. Photodetector 1706 is preferably one or more photodetectorsthat measure the intensity of light or one or more photodetectors thatimage the emitted light. Examples of photodetectors include cameras,photodiodes, avalanche photodiodes, CCD chips, CCD cameras,photomultiplier tubes, CMOS detectors, film, phosphorescent materials,and intensifiers. Photodetectors may be cooled to decrease backgroundsignals. In a preferred embodiment, photodetector 1706 is an array ofphotodiodes. In another preferred embodiment, photodetector 1706 is acharge coupled device (CCD) camera. Photodetector 1706 is connected tocomputer 1726 and electronics 1720. Photodetector 1706 may be joined tooptics 1708 and/or to light tight enclosure 1704. Photodetector 1706 mayalso incorporate control electronics, connectors and high speed cablesfor efficient transfer of images to electronics 1720 and computer 1726.The active surface of photodetector 1706 (or the imaging surface whenphotodetector 1706 is an imaging detector such as a CMOS or CCD chip) ispreferably matched to the size of the object (e.g., individual well,multi-well assay plate sector or multi-well assay plate) being imaged soas to balance the requirements for light capturing efficiency and thespatial resolution of the recorded image with the cost and size of thedetector (and associated optics). Preferably, the area of the activesurface or imaging surface of the photodetector is 25% to 200% of thearea being detected or imaged or more preferably between 50% and 100%.In a preferred embodiment of an imaging detector adapted to image astandard multi-well assay plate in six square sectors, the area of theimaging detector (e.g., a CCD or CMOS chip) is between 0.95 sq. in. and2.0 sq. in. or more preferably between 0.95 and 1.2 sq. in. In analternate embodiment, a smaller imaging detector may be used withoutsignificant loss in light capturing efficiency or resolution byincluding a tapered fiber optic bundle in optics 1708. For example,optics 1708 may include a combination of a lens, preferably atelecentric lens, that projects an image having an area of preferablybetween 25% and 100% (more preferably, between 50% and 100%) of the areabeing imaged and a tapered fiber optic bundle to reduce this image tothe size of the imaging detector.

Optics 1708 generally collect light emitted from multi-well assay plate1710 and focus that light on photodetector 1706. Optics 1708 mayinclude, for example, elements that transmit, scatter, block, filter,modify, diffract, refract, and/or reflect light. Optics 1708 may alsoinclude physical/mechanical elements that provide structural support orcouple the optical elements to other elements of reader 1700. Examplesof elements include lenses, prisms, filters, splitters, mirrors, opticalfibers, optical couplers, optical epoxies and adhesives, windows,modulators, optical coatings and the like. In a preferred embodiment,optics 1708 comprises a telecentric lens to achieve uniform collectionof light over a large area (which may otherwise be imaged in a distortedmanner by optics using non-telecentric lenses). The diameter of the lens(especially the front lens element of a multi-element lens) is,preferably, matched to the size of the object (e.g., multi-well assayplate sector) being imaged so as to balance the requirements for minimaldistortion and maximal light capturing efficiency with the cost of thelens. In a preferred embodiment of the lens adapted to image a standardmulti-well assay plate in six square sectors, the diameter of the lensor the first lens element in a compound lens is between 3.0″ and 5.0″ ormore preferably between 3.5″ and 4.5″ or most preferably between 3.9″and 4.3″. The lens, preferably, has a light capture efficiency ofgreater than 2% or more preferably, greater than 5% for hemisphericalradiation from point sources in the object plane. The full cone anglefor accepted light from the object plane is, preferably greater than10%, more preferably greater than 15%, even more preferably greater than20%, even more preferably greater than 25% or, most preferably, greaterthan 30%. In another embodiment, optics 1708 comprises one or moreoptical fibers or an optical fiber array. In another preferredembodiment, optics 1708 comprise a window and/or a filter and do notfocus light on photodetector 1706. In another embodiment, optics 1708comprise a lens and fiber optic bundle (e.g. a tapered fiber opticbundle). Optics 1708 may comprise a compliant coupling that allowsfocusing without disrupting the light tight properties of the connectionbetween optics 1708 and light tight enclosure 1704. Optics 1708 mayoptionally include filters designed to maximize the collection of adesired luminescent signal relative to background light. In a preferredembodiment, optics 1708 includes filters designed to selectively passthe luminescence generated from transition metal labels, particularlyruthenium-tris-bipyridine labels. Preferably, the optics in such asystem would block light the majority of light with a wavelength greaterthan 800 nm (or, more preferably, 750 nm) and optionally light with awavelength less than 500 nm (or, more preferably, 550 nm). The filterelements may, optionally, be removable or replaceable. According to onepreferred embodiment, the filter has a band pass characteristic with along wavelength cutoff (50% transmission) of 750 nm+/−25 nm and a shortwavelength cutoff less than 550 nm and/or has an average pass bandtransmission greater than 80%. According to another embodiment, theapparatus comprises a filter covering the light detector(s) (e.g., adichroic, interference and/or absorbance filter). For example, the lightdetector may be an array of light detectors comprising an array ofsilicon photodiodes covered by filters (e.g., dichroic, interferenceand/or absorbance filters).

Plate transport mechanism 1716 moves multi-well assay plates into,within and out of reader 1700. Plate transport mechanism 1716 comprisesa plate carrier 1740 that holds the multi-well assay plates duringtransport, one or more linear translation stages that move the platecarrier 1740, one or more magnetizable (preferably, reversiblymagnetizable) tabs, sensors, and a variety of mechanisms that alignand/or hold the multi-well assay plate to the carrier. Plate transportmechanism 1716 is primarily composed of metal and plastic. In oneembodiment, plate transport mechanism 1716 moves plates 1710 from platestacker 1736 through aperture 1714 into light tight enclosure 1704 andvisa versa. In an example of operation, one or more multi-well assayplates are loaded into plate stacker 1736. Under computer control, platetransport mechanism 1716 and an elevator in plate stacker 1736 are movedto the home position, which is verified by sensors. Plate transportmechanism 1716 is translated out of light tight enclosure 1704 throughaperture 1714 into plate stacker 1736. The movement of plate transportmechanism 1716 brings plate carrier 1740 into contact with elements thatretract a spring loaded rear slider and rotates a spring loadedpositioning element located on plate carrier 1740, readying platecarrier 1740 to receive a multi-well assay plate. An elevator in platestacker 1736, driven by a motor, raises the stack of plates. A springloaded latch in stacker 1736 is opened by a solenoid, allowing theelevator in plate stacker 1736 to lower the stack of plates until oneplate 1710 (on the bottom of the stack) has passed through the latch.The spring loaded latch then closes, and the stacker continues to lowerthe plate 1710 until plate 1710 is placed in the plate carrier 1740 ofplate transport mechanism 1716. A sensor, preferably an infrared sensor,verifies that plate 1710 is on plate carrier 1740. As the platetransport mechanism 1716 moves plate carrier 1740 out of plate stacker1736, the spring loaded positioning element releases and pushes plate1710 to register it against one side of plate carrier 1740. The springloaded rear slider also releases, covers part of the rear lip of plate1710 and pushes plate 1710 against another side of plate carrier 1740.Optionally, plate transport mechanism 1716 retracts plate carrier 1740,which actuates a pin that holds plate 1710 tightly to the plate carrier1740 such that upward vertical force applied to the bottom of plate 1710(for example, in an attempt to make good electrical contact withelectrical connector 1724) does not move the plate. Plate transportmechanism 1716 moves plate carrier 1740 through aperture 1714 into lighttight enclosure 1704. Aperture 1714 closes and plate transport mechanism1716 translates plate 1710 to bar code reader 1718, which identifiesplate 1710. Plate 1710 is translated until the first sector of plate1710 is aligned with optics 1708 and plate electrical connector 1724.After one or more electrochemiluminescent assay measurements areconducted, plate transport mechanism 1716 then removes plate 1710 fromlight tight enclosure 1704 by using a similar set of steps that may beconducted in a different order. In another embodiment, individual plates1710 are placed in plate carrier 1740 (for example, manually or byrobotics 1738). The motion of plate carrier 1740 is accomplished by oneor more linear actuators. In one embodiment, the actuators are drivenwith a stepper motor in an open loop configuration. The plate is movedto specific locations when computer 1726 instructs the stepper motor tomove a specified number of steps. In another embodiment, the motion ofplate carrier 1740 in plate transport mechanism 1716 is driven by DCmotors using a closed feedback loop controlled by computer 1726.

The movement and position of plate 1710 in plate carrier 1740 isverified by plate alignment mechanism 1712. Plate alignment mechanism1712 uses one or more sensors to verify certain positions of platecarrier 1740 and/or to set a reference point for its position. Thesensors can be, for example, mechanical sensors, optical sensors,electrical sensors, magnetic sensors or other sensors known for sensingposition of an object accurately. In a preferred embodiment, platealignment mechanism 1712 incorporates a Hall effect sensor that sensesone or more magnetizable (preferably, reversibly magnetizable) tabs(made, for example, from magnetizable steel) on plate carrier 1740 or onone or more axis of plate transport mechanism 1716 (the tab being sensedwhen it travels in between the Hall sensor and a magnet mounted oppositethe Hall sensor, thus blocking the effect of the magnet on the sensor).The tab and Hall sensor may be used to detect when plate transportmechanism 1716 is in the “home” position and may thus be used todetermine the true position of plate transport mechanism 1716. Inanother preferred embodiment, plate alignment mechanism incorporates aninfrared sensor that senses the interruption of light between aninfrared light source and an infrared light detector when plate 1710and/or plate carrier 1740 interrupt the path of the infrared light.Plate alignment mechanism 1712 may also include a sensor that verifiesthat the stepper motor has conducted a specified number of steps and/orto verify that the stepper motor has not stalled. In a preferredembodiment, this sensor comprises an optical encoder. In anotherpreferred embodiment, plate alignment mechanism 1712 incorporates apressure switch to detect the corner chamfer of a plate (e.g., thechamfers on the top and bottom left corners of plate top 932 in FIG.9A). The presence or absence of the chamfer determines the orientationof the plate in plate carrier 1740. If the sensor determines that theplate is in the incorrect orientation, computer 1726 may instruct theinstrument to stop the run, skip the plate or, more preferably, to readthe plate but to transpose the data so as to correct for themis-orientation (thus preventing costly delays or loss of precioussamples).

Bar code reader 1718 is used in the reader 1700 to identify specificmulti-well assay plates. The bar code reader is preferably a fixedposition laser bar code scanner, for example, an Opticon Series NLB9625/9645. Electronics 1720 participate in the operation, controlling,and monitoring of one or more electronic and/or mechanical elements inreader 1700. Electronics 1720 may comprise a variety of componentstypically encountered in devices, for example, wires, circuits, computerchips, memory, logic, analog electronics, shielding, controllers,transformers, I/O devices, and the like. Current/voltage source 1722 isan electrical circuit capable of generating defined voltage waveformsand/or defined current waveforms. Current/voltage source 1722 isconnected to electronics 1720, computer 1726 and plate electricalconnector 1724. In one embodiment of the invention, current/voltagesource 1722 includes a potentiostat. The potentiostat is advantageousfor reading plates that include independent reference electrodes andallows the potentials at the working and/or counter electrodes to bereferenced relative to the potential at the reference electrode.

Plate electrical connector 1724 makes contact with multi-well assayplate 1710 to allow the application of current and/or voltage waveformsby current/voltage source 1722. Plate electrical connector 1724comprises one or more connectors, electrical connections, a linearactuator and, optionally, a support. In a preferred embodiment, theconnectors are spring loaded to improve electrical contact with plate1710. Connectors may be made of any suitable material that has aconducting outer surface. Preferably, they are sufficiently durable towithstand repeatedly making contact with plates. Typically, theconnectors are comprised of a hard metal or metal alloy coated with ahighly conducting metal film (e.g. gold or silver). In a preferredembodiment, connectors include a waffle-point contact head comprised ofgold plated nickel/silver, spring loaded on a gold plated stainlesssteel spring in a nickel/silver receptacle, for example, connectorsoffered by Interconnect Devices, Inc. (GSS-18.3.8-G). In an alternativeembodiment, connectors are comprised of a compliant material coated witha highly conducting material. The support for the connectors may becomprised of any material that can support the connectors when theconnectors are pushed against plates. In a preferred embodiment, thesupport in plate electrical connector 1724 is comprised of a circuitboard, preferably with traces that electrically connect the connectorsto current/voltage source 1722 and/or electronics 1720. Plate electricalconnector 1724 may include a sensor (in a preferred embodiment, a Hallsensor) that verifies the home position. Plate electrical connector 1724may also incorporate a thermal sensor (e.g., a thermister, athermocouple, a platinum RTD), which in a preferred embodiment, isspring loaded on the support of plate electrical connector 1724. In oneembodiment, the thermal sensor makes contact with a multi-well assayplate 1710 to measure its temperature. The linear actuator in plateelectrical connector 1724 pushes the connectors (and optionally thesupport) into plate 1710 to make electrical connections.

Advantageously, the apparatus includes a temperature sensor orthermometer adapted to measure the temperature of a plate. Preferably,the temperature sensor or thermal sensor can detect the well temperaturewithin 5° C., more preferably within 3° C., even more preferably within1° C. and most preferred within 0.25° C. Even more preferably, thetemperature sensor can reach steady state within ten seconds, preferablywithin five seconds, even more preferably within three seconds. Thesensor may be a contact sensor (e.g., a thermister, a thermocouple, or aplatinum RTD). Alternatively it may be a non-contact-sensor such as anIR sensor. In a preferred embodiment, the apparatus comprises one ormore non-contact temperature sensors and the apparatus is adapted to beable to measure the temperature of various locations on the plate (e.g.,through the use of multiple sensors and/or by moving the plate relativeto the sensors). In another preferred embodiment, the apparatus furthercomprises a computer adapted to receive the signal from a temperaturesensor, report the temperature to the user and, preferably, adjust themeasured luminescence signals to account for the effects of temperatureon luminescent signals, electrochemiluminescent signals, and/or otherreactions occurring during the conduct of an assay. The computer,preferably, further comprises memory for saving data and/or calibrationcurves from calibration measurements conducted at a variety oftemperatures and software for using said data and/or calibration curvesto normalize test data against variations in temperature. According toanother embodiment, the apparatus also comprises a temperaturecontroller to control the temperature within the well. According to yetanother embodiment, the apparatus is adapted to reject or otherwise flagan assay plate (e.g., with an indication of a software error or warningor the like) if the temperature detected is outside a specified range.

Computer 1726 participates in the operation, controlling, managing ofdata, and monitoring of reader 1700 and/or other peripheral devices. Itis preferably comprised of a computer, a display, user input devices,data storage devices, I/O devices, networking devices, ethernetconnections, modems, optical connections, software and the like. Powersupply 1728 supplies electrical power to reader 1700 and/or otherdevices. Data and network connections 1730 may comprise connections,hardware, buses and the like. Data and network connections 1730 may be,for example, RS-232 ports, USB ports, PCMCIA cards, PCI boards, ethernetcards, modems and the like. Indicators 1732 provide information on theoperation and/or status of reader 1700 and may be, for example, lights,gauges, audible devices or devices that send and/or receive signalto/from computer 1726.

Reagent handler 1734 is one or more devices that add or remove reagentsto multi-well assay plates. In a preferred embodiment, reagent handler1734 is a pipetting station. Robotics 1738 may comprise one or moreelectromechanical devices that transport, incubate and/or mix multi-wellassay plates and the contents of their wells. Plate stacker 1736comprises one or more containers for holding one or more multi-wellassay plates and, advantageously, electrical and/or mechanical systemsfor moving plates. Plate stackers may also comprise mechanisms such aslatches, positioning elements, sliders, grabbers, push arms, etc., thatcan be used to control the movement and position of plates. Platestackers may have features that aid in the alignment and/or orientationof plates. Many plate stackers pre known in the art.

In the use of reader 1700, one or more multi-well assay platescontaining assay reagents in one or more wells are loaded into the inputstack of plate stacker 1736. (All of the following steps are undercontrol of computer 1726 and electronics 1720.) Plate stacker 1736 andplate transport mechanism 1716 move a multi-well assay plate 1710 fromthe input stack of plate stacker 1736 into plate carrier 1740, transportplate 1710 through input aperture 1714 and into light tight enclosure1704 as described above. Aperture 1714 closes and plate transportmechanism 1716 translates plate 1710 to bar code reader 1718, whichidentifies plate 1710. Plate 1710 is translated until the first sectorof plate 1710 is aligned with optics 1708 and plate electrical connector1724. Photodetector 1706 acquires and, preferably, stores a backgroundimage and sends data to computer 1726. Plate electrical connector 1724pushes against multi-well assay plate 1710 to bring the contacts ofelectrical connector 1724 into electrical contact with the first sectorof plate 1710. Photodetector 1706 begins to acquire an image andcurrent/voltage source 1722 generates a waveform that is applied toplate 1710 by plate electrical connector 1724. After completion of thewaveform and image, the data are transferred from photodetector 1706 andelectronics 1720 to computer 1726 where they are processed. Plateelectrical connector 1724 lowers away from plate 1710 to breakelectrical contact; a sensor verifies when plate electrical connector1724 is fully lowered. Plate transport mechanism 1716 translates plate1710 so that the next sector (if another sector is to be measured)becomes aligned with optics 1708 and plate electrical connector 1724 andthe process of making contact and acquiring a measurement are repeated.Reader 1700 continues to repeat these steps until all desiredmeasurements have been completed. Alternatively, more than one sectormay be contacted, fired and/or read at a time. In another alternateembodiment, the entire plate is fired and read at the same time. Afterthe final measurement, plate electrical connector 1724 is lowered andoutput aperture 1714 is opened. Plate transport mechanism 1716translates plate 1710 out of light tight enclosure 1704 through outputaperture 1714 and into the output stack of plate stacker 1736. Themovement of plate transport mechanism 1716 brings plate carrier 1740into contact with elements that retract a spring loaded rear slider androtates a spring loaded positioning element located on plate carrier1740, readying plate 1710 to be removed from plate carrier 1740. Platetransport mechanism 1716 and plate stacker 1736 move plate 1710 fromplate carrier 1740 to the output stack of output stacker 1736. A sensor,preferably an infrared sensor, verifies that plate 1710 is out of platecarrier 1740. Plate transport mechanism 1716 translates plate carrier1740 out of plate stacker 1736, through output aperture 1714 into lighttight enclosure 1704 and into home position. If desired, the processrepeats to read another plate.

In another embodiment of the use of reader 1700, robotics 1738 are usedto introduce plates into the input stack of plate stacker 1736. Whenmeasurements from a given multi-well assay plate are complete, it isreturned to plate stacker 1736 and removed by robotics 1738.

In some embodiments of reader 1700, one or more of cover 1702, optics1708, multi-well assay plate 1710, bar code reader 1718, data andnetwork connections 1730, indicators 1732, reagent handler 1734, platestacker 1736 and/or robotics 1738 may be omitted. In another embodimentof reader 1700, bar code reader 1718 is replaced with another device foridentifying plates, for example, a scanner, a camera, a magnetic stripreader, or the like. In another embodiment of reader 1700, one or morecomponents such as computer 1726, power supply 1728, data and networkconnections 1730, reagent handler 1734, plate stacker 1736 and/orrobotics 1738 are positioned inside cover 1702.

In another embodiment of reader 1700, a plurality of light tightenclosures 1704, photodetectors 1706, optics 1708, plate alignmentmechanisms 1712, plate transport mechanisms 1716, bar code readers 1718,electronics 1720, current/voltage sources 1722, plate electricalconnectors 1724, plate stacker 1736 and/or robotics 1738 are combinedwithin a single reader to provide additional capabilities such asimproved speed, throughput and efficiency.

FIG. 18 shows a preferred embodiment of reader 1700 in which selectedelements of reader 1800 are illustrated. Reader 1800 illustrates a lighttight enclosure 1804, photodetector 1806, optics 1808, plate transportmechanism 1816, plate electronics 1820, input plate stacker 1836A,output plate stacker 1836B, input plate stack 1837A, output plate stack1837B, and output door and/or aperture 1814B. Preferably photodetector1806 comprises a cooled CCD camera and optics 1808 comprise atelecentric lens. Plate stacks 1837A and 1837B can preferably holdbetween 1 and 50 96-well plates and between 1 and 75 384-well plates.

FIG. 19 illustrates selected elements of another embodiment of reader1700. Light tight enclosure 1904 is coupled to optics 1908, whichcomprise a lens and a filter (e.g., a filter designed to selectivelypass luminescence from ruthenium-tris-bipyridine labels). Optics 1908 iscoupled to photodetector 1906 which, preferably, comprises a CCD chip1907. Door and/or aperture 1914 is present as part of light tightenclosure 1904. Plate 1910, with sectors 1910A, 1910B, and 1910C, isheld in plate carrier 1940 attached to plate transport mechanism 1916.Plate electrical connector 1924 moves plate electrical connectorcontacts 1925 up and down to make and break contact, respectively, withcontact surfaces in a sector of plate 1910. In the position illustratedin FIG. 19, connector contacts 1925 are in electrical contact withsector 1910A of plate 1910. Plate transport mechanism 1916, togetherwith plate alignment mechanism (not illustrated) have aligned plate1910, and in particular, sector 1910A appropriately with optics 1908,plate electrical connector 1924 and plate electrical connector contacts1925. In another embodiment, plate electrical connector contacts 1925are not in contact with plate 1910, and plate transport mechanism 1916can translate plate 1910 such that another sector (e.g., sector 1910B or1910C) are aligned with optics 1908 and plate electrical connector 1924and plate electrical connector contacts 1925. Plate carrier 1940,preferably, holds plate 1910 such that plate 1910 resists the upwardforce exerted by plate electrical connector allowing plate electricalconnector contacts 1925 to apply sufficient pressure to plate contactson plate 1910 to achieve electrical contact with low contact resistance.In a preferred embodiment, this contact resistance is less than 10 ohms.In another preferred embodiment, the contact resistance is less than 10ohms, preferably less than 5 ohms, more preferably less than 2 ohms,even more preferably less than 1 ohm and most preferred less than 0.5ohms.

FIG. 20 illustrates selected elements of another embodiment of reader1700. Photodetector 2056 with imaging element 2057 is coupled to optics2058 comprising a telecentric lens and a filter element 2059. Multi-wellassay plate 2042, with sectors 2042A, 2042B, 2042C, 2042D, 2042B and2042F is held by plate carrier 2040 attached to plate transportmechanism 2047 (shown in part). In FIG. 20, optics 2058 collect an imageof sector 2042A and focus that image onto imaging element 2057 ofphotodetector 2056. In a preferred embodiment, sector 2042A has an areaequivalent to ⅙ the area of a standard 96-well microplate and optics2058 and imaging element 2057 have dimensions appropriate for imagingsuch a sector. In an especially preferred embodiment, optics 2058 is atelecentric lens with a diameter of approximately 4.1″ and imagingelement 2057 is a CCD chip with dimensions of approximately 1 inch by 1inch. Preferably, optics 2058 collect light from sector 2042A uniformlyand with reasonable efficiency. Plate transport mechanism 2047 cantranslate plate 2042 such that another sector (e.g. sector 2042B, etc.)is aligned with optics 1908.

In another embodiment of FIG. 17, reader 1700 comprises a cover 1702, alight tight enclosure 1704 with a door and/or aperture 1714, aphotodetector 1706, optics 1708, multi-well assay plate 1710, platealignment mechanism 1712, plate transport mechanism 1716, electronics1720, current/voltage source 1722, plate electrical connector 1724,computer 1726, power supply 1728, data and network connections 1730,indicators 1732, reagent handler 1734, one or more plate stacker 1736,plate carrier 1740 and robotics 1738.

Photodetector 1706 is preferably an array of photodiodes, and morepreferably, a linear array of eight photodiodes spaced to align with theeight wells in a row of wells in a 96-well plate. Photodetector 1706further comprises a circuit board on which the photodiodes are mounted.The photodiodes of photodetector 1706 preferably have a conductiveshield (most preferably made of brass) to reduce EMI. The photodiodeprinted circuit board preferably resides in metal case (e.g., analuminum case) to reduce EMI. Optics 1708 preferably comprise an opticalfilter and/or optical coating, and a thin shield to reduce opticalcrosstalk and the measurement of background or non-specific lightsignals. In a preferred embodiment, the light detector is an array oflight detectors comprising an array of photodiodes covered by dichroic,interference and/or absorbance filters (the filters, preferably, beingdesigned to exclude infra red light, most preferably light with a wavelength greater than 750 nm and, optionally, light with a wave lengthshorter than 550 nm).

During a measurement, photodetector 1706 and optics 1708 are in closeproximity to multi-well assay plate 1710.

Light tight enclosure 1704 is a sealed compartment designed to preventthe entrance or exit of light. Aperture 1714 incorporates a door thatopens to allow transport of multi-well assay plates into and out of thelight tight enclosure. The door opens and closes by sliding along atongue and groove configuration at the junction between the door andaperture 1714 that provides a tortuous path that reduces transmission oflight. The movement of the door in aperture 1714 is mechanically drivenby an actuator (e.g., a linear actuator and/or a belt driven by a motorsuch as a stepper motor) that is controlled by computer 1726 andelectronics 1720. The door in aperture 1714 can also be activated bypressing a touch button. Light tight enclosure 1704 enclosesphotodetector 1706, plate carrier 1740, plate 1710 and the connectorcontacts of electrical contact mechanism 1724. The walls of the lighttight enclosure are preferably black to reduce reflection of light.

Plate transport mechanism 1716 moves multi-well assay plates within thereader 1700. Plate transport mechanism comprises a plate carrier 1740that holds the multi-well assay plates during transport, a lineartranslation stage that move the plate carrier 1740, one or moremagnetizable (preferably, reversibly magnetizable) tabs, sensors, and avariety of mechanisms that align and/or hold the multi-well assay plateto the carrier. Plate transport mechanism 1716 translates plate carrier1740 along a single axis within light tight enclosure 1704. Plate 1710is translated so that a sector of plate 1710 can be aligned withphotodetector 1706 and plate electrical connector 1724. The motion ofplate carrier 1740 is accomplished by an actuator (e.g., a linearactuator and or a belt driven by a motor such as a stepper motor)located outside the light tight enclosure 1704. In one embodiment, theactuators are driven with a stepper motor in an open loop configuration.The plate is moved to specific locations when computer 1726 instructsthe stepper motor to move a specified number of steps. In anotherembodiment, the motion of plate carrier 1740 in plate transportmechanism 1716 is driven by DC motors using a closed feedback loopcontrolled by computer 1726. Individual plates 1710 are placed in platecarrier 1740 (for example, manually or by robotics 1738).

The movement and position of plate 1710 in plate carrier 1740 isverified by plate alignment mechanism 1712. Plate alignment mechanism1712 incorporates a Hall effect sensor that verifies certain positionsof plate carrier 1740 and/or sets a reference point for its position(i.e., by sensing one or more magnetizable (preferably, reversiblymagnetizable) tabs (made, for example, from magnetizable steel) on platecarrier 1740 or on one or more axis of plate transport mechanism 1716(the tab being sensed when it travels in between the Hall sensor and amagnet mounted opposite the Hall sensor, thus blocking the effect of themagnet on the sensor). Alternatively, plate alignment mechanism 1712incorporates an infrared sensor that senses the interruption of lightbetween an infrared light source and an infrared light detector whenplate 1710 and/or plate carrier 1740 interrupt the path of the infraredlight. Plate alignment mechanism 1712 may also include a sensor thatverifies that the stepper motor has conducted a specified number ofsteps and/or to verify that the stepper motor has not stalled. In apreferred embodiment, this sensor comprises an optical encoder. In apreferred embodiment, plate alignment mechanism 1712 incorporates apressure switch to detect the corner chamfer of a plate (e.g., thechamfers on the top and bottom left corners of plate top 932 in FIG.9A). The presence or absence of the chamfer determines the orientationof the plate in plate carrier 1740. If the sensor determines that theplate is in the incorrect orientation, computer 1726 may instruct theinstrument to stop the run, skip the plate or, more preferably, to readthe plate but to transpose the data so as to correct for themis-orientation (thus preventing costly delays or loss of precioussamples).

Electronics 1720 participate in the operation, controlling, andmonitoring of one or more electronic and/or mechanical elements inreader 1700. Electronics 1720 may comprise a variety of componentstypically encountered in devices, for example, wires, circuits, computerchips, memory, logic, analog electronics, shielding, controllers,transformers, I/O devices, and the like. Current/voltage source 1722 isan electrical circuit capable of generating defined voltage waveformsand/or defined current waveforms. Current/voltage source 1722 isconnected to electronics 1720, computer 1726 and plate electricalconnector 1724. In one embodiment of the invention, current/voltagesource 1722 includes a potentiostat. The potentiostat is advantageousfor reading plates that include independent reference electrodes andallows the potentials at the working and/or counter electrodes to bereferenced relative to the potential at the reference electrode.

Plate electrical connector 1724 makes contact with multi-well assayplate 1710 to allow the application of current and/or voltage waveformsby current/voltage source 1722. Plate electrical connector 1724,preferably, comprises one or more connector contacts, electricalconnections, a linear actuator and, optionally, a support. In apreferred embodiment, the connector contacts are spring loaded toimprove electrical contact with plate 1710. Connector contacts may bemade of any suitable material that has a conducting outer surface.Preferably, they are sufficiently durable to withstand repeatedly makingcontact with plates. Typically, connector contacts are comprised of ahard metal or metal alloy coated with a highly conducting metal film(e.g. gold or silver). In a preferred embodiment, connector contacts area waffle-point contact head comprised of gold plated nickel/silver,spring loaded on a gold plated stainless steel spring in a nickel/silverreceptacle, for example, contacts offered by Interconnect Devices, Inc.(GSS-18.3.8-G). In an alternative embodiment, connector contacts arecomprised of a compliant material coated with a highly conductingmaterial. The support for the connector contacts may be comprised of anymaterial that can support the connector contacts when the contacts arepushed against plates. In a preferred embodiment, the support in plateelectrical connector 1724 is comprised of a circuit board, preferablywith traces that electrically connect the contacts to current/voltagesource 1722 and/or electronics 1720. Plate electrical connector mayinclude a sensor (in a preferred embodiment, a Hall sensor) thatverifies the home position. Plate electrical connector 1724 may alsoincorporate a thermal sensor (e.g., a thermister, a thermocouple, aplatinum RTD), which in a preferred embodiment, is spring loaded on thesupport of plate electrical connector 1724. In one embodiment, thethermal sensor makes contact with a multi-well assay plate 1710 tomeasure its temperature. The linear actuator in plate electricalconnector 1724 pushes the connector contacts (and optionally thesupport) into plate 1710 to make electrical connections. In a preferredembodiment, plate electrical connector 1724 has seven electricalconnector contacts arranged in a line. In this embodiment between oneand six working connector contacts may contact contact surfacesconnected to working electrodes on plate 1710 and between one and sixcounter connector contacts may contact contact surfaces connected to thecounter electrodes on plate 1710.

Advantageously, the apparatus includes a temperature sensor orthermometer adapted to measure the temperature of a plate. Preferably,the temperature sensor or thermal sensor can detect the well temperaturewithin 5° C., more preferably within 2° C., even more preferably within1° C. and most preferably within 0.25° C. Even more preferably, thetemperature sensor can reach steady state within ten seconds, preferablywithin five seconds, even more preferably within three seconds. Thesensor may be a contact sensor (e.g., a thermister, a thermocouple, or aplatinum RTD). Alternatively it may be a non-contact sensor such as anIR sensor. In a preferred embodiment, the apparatus comprises one ormore non-contact temperature sensors and the apparatus is adapted to beable to measure the temperature of various locations on the plate (e.g.,through the use of multiple sensors and/or by moving the plate relativeto the sensors). In another preferred embodiment, the apparatus furthercomprises a computer adapted to receive the signal from a temperaturesensor, report the temperature to the user and, preferably, adjust themeasured luminescence signals to account for the effects of temperatureon luminescent signals, electrochemiluminescent signals, and/or otherreactions occurring during the conduct of an assay. The computer,preferably, further comprises memory for saving data and/or calibrationcurves from calibration measurements conducted at a variety oftemperatures and software for using said data and/or calibration curvesto normalize test data against variations in temperature. According toanother embodiment, the apparatus also comprises a temperaturecontroller to control the temperature within the well.

In operation, case 1702 is opened and aperture 1714 is opened, either bycomputer 1726 or by pressing a touch button located on case 1702. Amulti-well assay plate is loaded into plate carrier 1740 which resideswithin light tight enclosure 1104, and aperture 1714 is closed. Undercontrol of computer 1726, plate transport mechanism translatesmulti-well assay plate 1710 until the first sector of plate 1710 isaligned with the photodiode array of photodetector 1706 and with thecontacts of plate electrical connector 1724. Photodiode array ofphotodetector 1706 is held in close proximity to the upper surface ofplate 1710 to improve the efficiency of optical collection and to reduceoptical crosstalk between wells. Plate electrical connector 1724 pushesthe connector contacts into plate 1710 to create an electricalconnection. Current/voltage source 1722 generates a waveform that isapplied to plate 1710 via plate electrical connector 1724. Photodetector1706 measures the light emitted from the active sector in plate 1710.Each of the eight photodiodes in photodetector 1706 are located about awell in a row of wells in the multi-well assay plate and the lightrecorded on a particular photodiode is identified as the light collectedfrom a particular well. Preferably, there is also software correction tocompensate for the expected amount of crosstalk due to light from a wellhitting a light detector aligned with a different well. Signal collectedby photodetector 1706 is sent to computer 1726. After the measurement iscomplete, plate electrical connector 1724 retracts the connectorcontacts from plate 1710, and plate transport mechanism 1716 translatesplate 1710 so that the next sector is aligned with plate electricalconnector 1724 and with the photodiode array of photodetector 1706.Contact with plate 1710 is resumed, and excitation/detection of lightoccurs again. This cycle is repeated until all desired measurements arecompleted. At the completion of measurements for a given plate 1710,plate transport mechanism 1716 translates plate carrier 1740 until plate1710 is aligned with the door in aperture 1714. Aperture 1714 opens andplate 1710 is removed.

FIG. 21 illustrates selected elements of an embodiment of FIG. 17.Photodetector 2106 comprises an array of photodiodes 2107 and a circuitboard 2105. Photodiode array 2107 comprises eight photodiodes arrangedin a line. Plate carrier 2140, attached to plate transport mechanism2116, holds plate 2110. Plate 2110, a multi-well assay plate of theinvention, has 12 sectors 2110A-L. In FIG. 21, sector 2110A of plate2110 is positioned below photodiode array 2107. Plate 2110 has 96 wells;each sector contains 8 wells arranged in a line. The photodiode array2107 is configured such that each of the eight wells in a sector ofplate 2110 can be located directly below a unique photodiode inphotodiode array 2107. The top of a sector of plate 2110 is held inclose proximity to photodiode array 2107 to improve the efficiency oflight collection and to reduce optical crosstalk between wells. In FIG.21, multi-well assay plate 2110 is also positioned so that the contactsfor sector 2110A are aligned with connector contacts 2125 of plateelectrical connector 2124. In operation, plate electrical connector 2124pushes connector contacts 2125 into the back side of sector 2110A ofplate 2110 to establish electrical contact. Plate carrier 2140,preferably, holds plate 2110 to resist the upward force imposed by plateelectrical connector 2124. If plate electrical connector 2124 hasconnector contacts 2125 retracted from plate 2110, plate transportmechanism 2116 can translate plate carrier 2140 so that another sector(e.g., sector 2110B) becomes aligned with plate electrical connector2124, connector contacts 2125 and the photodiodes 2107 of photodetector2106.

FIG. 22 illustrates selected elements of an embodiment of FIG. 17. Lighttight enclosure 2204 houses photodetector 2207, plate 2210, platecarrier 2240, a plurality of connector contacts 2205 of plate electricalconnector 2224 and shield 2208. Photodetector 2207 comprises an array ofphotodiodes, with individual photodiodes 2207A, 2207B, 2207C, 2207D,2207E, 2207F, 2207G, and 2207H. The shield 2208 is attached tophotodetector 2207 to prevent electromagnetic interference. The shield2208 is preferably made of a conductive material such a metal, mostpreferably brass. Plate 2210 comprises 96 individual wells; FIG. 22shows eight wells, 2210A, 2210B, 2210C, 2210D, 2210E, 2210F, 2210G, and2210H that comprise one sector of plate 2210. Plate 2210 is held bycarrier 2240. Plate electrical connector 2224 pushes connector contacts2205 into the bottom of plate 2210 to establish electrical connectionsto one sector of plate 2210. Plate carrier 2240 positions plate 2210 sothat the sector of plate 2210 is aligned with connector contacts 2205and with photodetector 2207. The position of plate 2210 is such thatwell 2210A is aligned directly with photodiode 2207A; well 2210B isaligned with photodiode 2207B, and so on. Connector contacts 2205 arelined up with the bottom of the wells to contact seven walls between theeight wells of a row. Light emitted from each well is collectedprimarily by its corresponding photodiode. Preferably, there is alsosoftware correction of the signal received by the photodiodes, thecorrection compensating for the expected amount of crosstalk due tolight from a well hitting a light detector aligned with a differentwell.

FIG. 23 illustrates selected elements of a preferred embodiment of FIG.17. Reader 2300 includes a chassis 2301, photodetector 2306, multiwellassay plate 2310, plate transport mechanism 2316, plate electricalconnector 2324 and a plurality of connector contacts 2325. Photodetector2306 preferably comprises a plurality of photodiodes, a photodetectorcircuit board, a shield and a metal cover (shown in FIG. 23). Otherelements of reader 2300 are not shown in FIG. 23.

In further embodiments of FIG. 17, reader 1700 may measure the lightemitted by light emitting substances other than electrochemiluminescentlabels. For example, reader 1700 may be used for fluorescence assays,chemiluminescence assays, radioactive assays employing light emittingscintillants, bioluminescence assays, etc. It may also be used forabsorbance and scattering based measurements. In one embodiment, optics1708 further comprises one or more light sources and appropriate opticalelements for stimulating and detecting fluorescent labels. In anotherembodiment, optics 1708 further comprises one or more light sources andappropriate optical elements for absorbance or scattering measurements.In another embodiment, reagent handler 1734, optics 1708 andphotodetector 1706 further comprises appropriate reagent handlingequipment for chemiluminescent or bioluminescent assays. For example,some chemiluminescent assays require measurement of chemiluminescencesignals after a short and controlled time after addition of achemiluminescent reagent, so it is advantageous to include within theapparatus plate washers and/or means for dispensing reagents in acontrolled manner. Such dispensing means may include pipettes, syringesor other fluid dispensers adapted to deliver fluid to one well at a timeor multiple wells at a time. In operation, a plate is introduced intothe instrument, the plate is optionally washed by an integrated platewasher, a chemiluminescence reagent is optionally introduced by anintegrated fluid dispenser and the chemiluminescence is monitored(optionally after incubating the plate for a controlled period of timeafter washing or introduction of reagents).

5.4 Methods of Measuring Luminescence

Another aspect of the invention relates to methods for measuringluminescence from an assay plate, preferably a multi-well assay platehaving a plurality of wells. Preferably, the multi-well plate has astandard plate configuration such as a 96-well, 384-well plate, etc.

One preferred embodiment of the invention relates to methods ofmeasuring luminescence using any of the apparatuses and/or assay platesdescribed above.

The method comprises measuring luminescence, preferably electrodeinduced luminescence, more preferably electrochemiluminescence, emittedfrom the wells or assay domains. In the case ofelectrochemiluminescence, the method may also comprise providingelectrical energy to the plurality of wells or assay domains orotherwise inducing luminescence. In the case of fluorescence, forexample, the method may comprise inducing fluorescence by directing alight source onto an assay region. In the case of chemiluminescence, themethod may comprise adding a chemical initiator to the assay region.

According to one embodiment, the method involves measuring theluminescence in sectors. According to another embodiment, the methodincludes providing electrical energy to the multi-well assay plate insectors. As described above, measuring the plate in sectors providesimproved luminescence collection efficiencies. Moreover, it allows forthe use of a smaller imaging surface and/or the use of a smaller numberof light detectors.

Accordingly, a preferred embodiment of the invention relates to a methodfor measuring luminescence from a multi-well assay plate having aplurality of independently addressable sectors of jointly addressablewells, the method comprising:

-   -   (a) providing electrical energy to the multi-well assay plate;        and    -   (b) measuring luminescence from the multi-well assay plate in        sectors.

Another embodiment relates to a method for measuring luminescence from amulti-well assay plate having a plurality of independently addressablesectors of jointly addressable wells, the method comprising:

-   -   (a) providing an electrical connection to the multi-well assay        plate in sectors; and    -   (b) measuring luminescence from the multi-well assay plate in        sectors.

Yet another embodiment relates to a method for measuring luminescencefrom a multi-well assay plate having a plurality of wells comprising:

-   -   (a) providing electrical energy to a first sector of the        plurality of wells;    -   (b) measuring luminescence from the first sector of the        plurality of wells;    -   (c) providing electrical energy to a second sector of the        plurality of wells; and    -   (d) measuring luminescence from the second sector of the        plurality of wells.

A still further embodiment of the invention relates to a method formeasuring luminescence from a multi-well assay plate having a pluralityof wells comprising:

-   -   (a) measuring luminescence from a first sector of the plurality        of wells; and    -   (b) measuring luminescence from a second sector of the plurality        of wells.

A still further embodiment relates to a method for measuringluminescence from a multi-well assay plate having a plurality of wellscomprising:

-   -   (a) providing electrical energy to a first sector of the        plurality of wells; and    -   (b) providing electrical energy to a second sector of the        plurality of wells.

A still further embodiment relates to a method of conducting one or moreassays using an apparatus for measuring luminescence from an assayplate, preferably a multi-well assay plate having an array of wells,comprising a substrate having a top surface and a bottom surface and theapparatus comprising a light detector adapted to measure luminescenceemitted from the assay regions or assay wells, wherein the plate is heldonto a measuring platform during the measuring luminescence and/orduring the inducing the luminescence, particularly if the electricalconnector contacts push up on the plate from the bottom. The term “heldonto” is intended to refer to holding the plate down as electricalconnectors are pressing against the plate. This is advantageous sinceeven slight movement of the plate can alter the light detection orimaging. The plate can be “held down” from the bottom (e.g.,magnetically), the top (e.g., securing devices come down into the plateedges) or the sides (e.g., the sides are clamped onto a support).

5.4.1 Imaging Methods

One embodiment of the invention relates to a method of conducting aluminescence assays employing imaging systems, preferably imagingsystems comprising a camera. More specifically, an imaging system whichimages the luminescence emitted from the assay plate or multi-wellplate.

One preferred embodiment of the invention relates to a method formeasuring luminescence from a multi-well plate having a plurality ofwells comprising simultaneously imaging emitted luminescence from atleast two of the plurality of wells, wherein the imaging collects a coneof luminescence having a cone full angle of at least 10 degrees,preferably at least degrees, more preferably at least 20 degrees, evenmore preferably at least 25 degrees and most preferably at least 30degrees.

Another aspect of the invention relates to methods for measuringluminescence from a multi-well assay plate comprising the step ofimaging the emitted luminescence in sectors.

One embodiment of the invention relates to a method for measuringluminescence from a multi-well assay plate having a plurality of wellscomprising:

-   -   (a) forming a first image of a first sector of the multi-well        assay plate with an imaging system; and    -   (b) forming a second image of a second sector of the multi-well        assay plate.

Another embodiment method for measuring luminescence from a multi-wellassay plate having a plurality of wells comprising:

-   -   (a) aligning a first sector of the multi-well assay plate with        an imaging system;    -   (b) measuring luminescence from the first sector of the        multi-well assay plate with the imaging system;    -   (c) aligning a second sector of the multi-well assay plate with        the imaging system; and    -   (d) measuring luminescence from the second sector of the        multi-well assay plate with the imaging system.

Another embodiment relates to a method for measuring luminescence from amulti-well assay plate having a plurality of wells comprising:

-   -   (a) providing electrical energy to a first sector of the        plurality of wells;    -   (b) measuring luminescence from the first sector of the        plurality of wells using an imaging system;    -   (c) providing electrical energy to a second sector of the        plurality of wells; and    -   (d) measuring luminescence from the second sector of the        plurality of wells using the imaging system.

Preferably, the method employing an imaging system employs an apparatusand/or an assay plate, preferably multi-well assay plate, as describedabove.

Another embodiment of the invention relates to a method comprisingintroducing approximately 25-300 micro liters of assay mixture into eachof the plurality of wells and measuring the assay mixture from thewells, more preferably 75-200, more preferably 125-175, even morepreferably approximately 150 micro liters of assay mixture into thewells of a 96 well plate. Another embodiment relates to introducing20-60 micro liters of assay mixture, preferably 30-40 micro liters, andeven more preferably approximately 35 micro liters, into each of theplurality of wells and measuring the assay mixture from the wells,preferably from the wells of a 384 well plate.

Another embodiment of the invention relates to a method of conductingone or more assays using an apparatus for measuring luminescence from amulti-well plate, the multi-well plate having a standard configurationof wells and comprising a substrate having a top surface and a bottomsurface, and the apparatus comprising a light detector adapted tomeasure luminescence emitted from the plurality of wells, wherein themethod comprises:

-   -   (a) contacting each sector of the bottom surface with a        plurality of electrical connector contacts at one or more sector        contact locations, wherein the plurality of electrical connector        contacts contact the bottom surface between the wells; and    -   (b) measuring emitted luminescence.

Preferably, the sector contact locations comprise one or more,preferably two or more, more preferably three or more, even morepreferably four or more, and most preferably all of the followinglocations shown in FIG. 34A or 34B and as discussed above in relation tonovel plate bottom configurations.

Preferably, the sector contact locations comprise one or more,preferably two or more, more preferably three or more, even morepreferably four or more and most preferably all of the followinglocations shown in FIG. 34A or 34B and discussed above in relation tonovel plate bottom configurations.

Another embodiment of the invention relates to a method of conductingone or more assays using an apparatus for measuring luminescence from amulti-well plate,

-   -   the multi-well plate comprising a substrate having a top surface        and a bottom surface, the multi-well plate having an array of        wells corresponding to a standard 96-well plate configuration,        the array comprising one or more preferably two or more, more        preferably all, of the following:        -   a first sector comprising wells A1 through A4, B1 through            B4, C1 through C4, and D1 though D4;        -   a second sector comprising wells A5 through A8, B5 through            B8, C5 through C8, and D5 though D8;        -   a third sector comprising wells A9 through A12, B9 through            B12, C9 through C12, and D9 through D12;        -   a fourth sector comprising wells E1 through E4, F1 through            F4, G1 through G4, and H1 though H4;        -   a fifth sector comprising wells E5 through E8, F5 through            F8, G5 through G8, and H5 though H8; and        -   a sixth sector comprising wells E9 through E12, F9 through            F1, G9 through G12, and H9 though H12 (each of the            designations referring to a region of the well defined by            the row and column);    -   the apparatus comprising a light detector adapted to measure        luminescence emitted from the plurality of wells,        -   wherein the method comprises:        -   (a) contacting each sector of the bottom surface with a            plurality of electrical connector contacts at one or more            sector contact locations, wherein the plurality of            electrical connector contacts contact the bottom surface            between the wells; and        -   (b) measuring emitted luminescence.

According to a preferred embodiment of the invention, each sector iscontacted by the plurality of electrical connector contacts at leasttwo, preferably at least three, more preferably at least four, even morepreferably at least five and most preferred at least six locations.

Preferably, each sector is contacted by a 2×3 array of locations.

According to another preferred embodiment, the sector contact locationscomprise one or more of the following locations:

-   -   (i) two or more, preferably three or more, more preferably four        or more, even more preferably five or more, and most preferred        all of first sector locations: A1-B2; A2-B3; A3-B4; C1-D2;        C2-D3; C3-D4;    -   (ii) two or more, preferably three or more, more preferably four        or more, even more preferably five or more, and most preferred        all of second sector locations: A5-B6; A6-B7; A7-B8; C5-D6;        C6-D7; C7-D8;    -   (iii) two or more, preferably three or more, more preferably        four or more, even more preferably five or more, and most        preferred all of third sector locations: A9-B10; A10-B11;        A11-B12; C9-D10; C10-D11; C11-D12;    -   (iv) two or more, preferably three or more, more preferably four        or more, even more preferably five or more, and most preferred        all of fourth sector locations: E1-F2; E2-F3; E3-F4; G1-H2;        G2-H3; G3-H4;    -   (v) two or more, preferably three or more, more preferably four        or more, even more preferably five or more, and most preferred        all of fifth sector locations: E5-F6; E6-F7; B7-F8; G5-H6;        G6-H7; G7-H8; and    -   (vi) two or more, preferably three or more, more preferably four        or more, even more preferably five or more, and most preferred        all of sixth sector locations: E9-F10; E10-F11; E11-F12; G9-H10;        G10-H11; G11-H12.

Another embodiment relates to a method of conducting one or more assaysusing an apparatus for measuring luminescence from a multi-well assayplate,

-   -   the multi-well plate comprising a substrate having a top surface        and a bottom surface, the multi-well plate having an array of        wells corresponding to a standard 384-well plate configuration,        the array comprising rows A through P and columns 1 through 24        (described above in relation to plate bottoms)    -   the apparatus comprising a plurality of electrical connector        contacts, wherein the plurality of electrical connector contacts        contact the bottom surface between wells, and a light detector        adapted to measure luminescence emitted from the plurality of        wells.

Preferably, each sector comprises one or more electrical contacts at oneor more (preferably all) of the following locations:

-   -   (i) two or more, preferably three or more, more preferably four        or more, even more preferably five or more, and most preferred        all of first sector locations: B2-C3; B4-C5; B6-C7; F2-G3;        F4-G5; F6-G7;    -   (ii) two or more, preferably three or more, more preferably four        or more, even more preferably five or more, and most preferred        all of second sector locations: B10-C11; B12-C13; B14-C15;        F10-G11; F12-G13; F14-G15;    -   (iii) two or more, preferably three or more, more preferably        four or more, even more preferably five or more, and most        preferred all of third sector locations: B18-C19; B20-C21;        B22-C23; F18-G19; F20-G21; F22-G23;    -   (iv) two or more, preferably three or more, more preferably four        or more, even more preferably five or more, and most preferred        all of fourth sector locations: J2-K3; J4-K5; J6-K7; N2-O3;        N4-O5; N6-O7;    -   (v) two or more, preferably three or more, more preferably four        or more, even more preferably five or more, and most preferred        all of fifth sector locations: J10-K11; J12-K13; J14-K15;        N10-O11; N12-O13; N14-O15; and    -   (vii) two or more, preferably three or more, more preferably        four or more, even more preferably five or more, and most        preferred all of sixth sector locations: J18-K19; J20-K21;        J22-K23; N18-O19; N20-O21; N22-O23.

5.4.2 Methods Employing Light Detector Arrays

Another aspect of the invention relates to methods for measuringluminescence using an array of light detectors comprising:

-   -   (a) providing electrical energy to a first sector of the        plurality of wells;    -   (b) measuring luminescence from the first sector of the        plurality of wells with an array of light detectors;    -   (c) providing electrical energy to a second sector of the        plurality of wells; and    -   (d) measuring luminescence from the second sector with the array        of light detectors.

Another embodiment includes a method for measuring luminescence from amulti-well assay plate having a plurality of wells comprising:

-   -   (a) providing electrical energy to a first sector of the        plurality of wells;    -   (b) measuring luminescence from the first sector of the        plurality of wells using an array of light detectors;    -   (c) providing electrical energy to a second sector of the        plurality of wells; and    -   (d) measuring luminescence from the second sector of the        plurality of wells using an array.

Yet another embodiment relates to a method comprising:

-   -   (a) providing electrical energy to a first sector of the        multi-well assay plate;    -   (b) measuring luminescence from the first sector of the        multi-well assay plate with an array of light detectors;    -   (c) aligning a second sector of the multi-well assay plate with        the light detector, and    -   (d) measuring luminescence from the second sector of the        multi-well assay plate with the array of light detectors.

Another embodiment includes a method for measuring luminescence from amulti-well assay plate having a plurality of wells comprising:

-   -   (a) providing electrical energy to a first sector of the        multi-well assay plate;    -   (b) measuring luminescence from the first sector of the        multi-well assay plate with an array of light detectors;    -   (c) aligning a second sector of the multi-well assay plate with        the array of light detectors; and    -   (d) measuring luminescence from the second sector of the        multi-well assay plate with the array of light detectors.

Another embodiment of the invention relates to methods of conducting oneor more assays using an apparatus for measuring luminescence from amulti-well assay plate having a plurality of wells arranged in an array,the method comprising inducing and measuring the luminescence from theplurality of wells row by row or column by column.

Yet another embodiment relates to a method comprising contacting themulti-well plate bottom with at least one counter electrode connectorcontact and at least one working electrode connector contact tosimultaneously induce luminescence in a row or column of wells,preferably at least two counter electrode connector contacts and atleast two working electrode connector contacts, more preferably at leastthree counter electrode connector contacts and at least three workingelectrode connector contacts, and most preferred three counter electrodeconnector contacts and four working electrode connector contacts.

Preferably, the light detector comprises one or more photodiodes, morepreferably an array of photodiodes.

Preferably, the measuring luminescence comprises measuring luminescencefrom each well using at least one light detector aligned with each wellbeing measured.

Preferably, less than 2% of luminescence is cross-talk luminescence,more preferably less than 1%, even more preferably less than 0.5%, andmost preferred less than 0.1%.

Another aspect of the invention relates to measuring luminescence fromdifferent plate formats using the same apparatus, particularly anapparatus having a fixed array of light detectors (e.g., a linear arrayof eight photodiodes). Using this aspect of the invention, an apparatusconfigured for one type of plate format (e.g., a 96-well plate) can beused with other plate formats (e.g., a 384-well plate) with little or nomodification to the apparatus. For example, apparatuses have beendescribed above as being adapted to read ECL from multi-well plates inthe 96-well format wherein an array of eight photodiodes reads ECLemitted from each column of wells with each photodiode corresponding toa single well at each measurement step (i.e., each column compriseseight wells (8 rows×12 columns); the measurement of the plate isperformed in twelve steps (one measurement/inducement step per column)).

Surprisingly, the same apparatus may be employed to read other platessuch as 384-well plates, 96-well plates with 4-spot wells and othermulti-spot plates with the same fixed array of light detectors, wherethe apparatus and/or plate is either configured as described above orwith minor modifications to the instrument and/or plate design.

One embodiment employs an apparatus having an array of light detectorssuch as an array of eight photodiodes where, without modifying theinstrument, two or more alternative plate formats can be measured.Preferably, the apparatus is adapted to measure an assay plate whereinthe number of times the array of light detectors is shifted to the nextrow or column of wells is less than the number of times ECL is induced(e.g., voltage is applied to the plate). For example, one methodinvolves moving the light detector array (with respect to the plate) 12times, wherein ECL is induced at least 24 times, preferably at least 48times, more preferably at least 84 times and most preferred at least 120times.

According to a still further embodiment of the invention, an apparatushaving an array of light detectors is used to measure luminescence frommulti-well plates wherein each well comprises a plurality of assay spotsor assay domains (See, FIG. 3A-3C). More specifically, for example, aplate having a plurality of assay domains within each well (e.g., 4-spotplate, FIG. 3A) can be made to work in an apparatus having a single rowof photodiodes (thus a single photodiode per well per column). Thisplate type could be a standard 96 well plate top with 4 independentlyaddressable spots for measuring 4 different analytes in each well. Sincethe 4 spot well plate requires that 4 spots be fired in sequence in asingle fluid volume, the working electrode is preferably sectioned intofour separate, addressable leads that are electrically isolated from oneanother. A single counter electrode could be connected together acrosseach row of plates as in the standard plate, i.e., a small portion ofcounter electrode on opposite edges of the well bottom (See, FIG. 3A,counter electrodes 306A and B).

For example, such an apparatus can be used to measure a 96-well platewherein each well comprises four discrete spots (“a 4-spot 96-wellplate”) in addition to measuring a single spot 96-well plate. Since eachphotodiode in the array corresponds to a single well during each step ofthe measurement scan, the photodiode would not be capable of measuringthe 4 spots simultaneously. Therefore, the 4 spots per well arepreferably fired sequentially. This may be achieved by indexing theplate using smaller distances than the size of the wells. That is,modifying the plate bottom to include independently addressable contactsand leads for each working electrode of each spot. Thus, for each singlecolumn of wells of the 96-well plate, the corresponding bottom wouldhave four working electrode contacts for each spot of the 4-spot wells.

Referring to FIG. 38, well 3810 comprises four assay domains on fourindependently addressable working electrode surfaces 3811, 3812, 3813and 3814, which are electrically connected to four independentlyaddressable working electrode contacts 3820, 3821, 3822 and 3823 viaworking electrode connections 3824, 3825, 3826 and 3827. Workingelectrode contacts 3820, 3821, 3822 and 3823 are each contacted by theapparatus (e.g., at locations “x”). Portions (e.g., portion 3860) of theworking electrode connections within well 3810 are preferably coveredwith dielectric (not shown) so that the only exposed electrode surfacewithin well 3810 when the working electrode contact (e.g., workingelectrode contact 3820) is contacted is the corresponding workingelectrode surface (e.g., working electrode surface 3812). Workingelectrode contacts 3820, 3821, 3822 and 3823 are also preferablyelectrically connected to four working electrodes within one or moreadjacent wells (not shown) via working electrode connectors 3840, 3841,3842 and 3843. Thus, referring to FIG. 34B, two adjacent wells on eachside of a contact location “x” can both be addressed using a single setof four contacts. By using multiple contacts “x” as shown in FIG. 34 B,all eight wells of a column can be similarly contacted so as to allowfour (or more) spots within the wells to be sequentially fired and thussequentially measured with a single linear array of eight lightdetectors.

Counter electrode 3850 is preferably contacted by the apparatus at fourlocations (*) and is preferably electrically connected to well 3810 viacounter electrode connections 3851A and/or 3851B to exposed counterelectrode surfaces 3854A and/or 3854B and/or counter electrode 3850 maybe partially exposed within well 3810 (e.g., exposed counter electrodesurface 3855). Counter electrode 3850 may also be electrically connectedto one or more adjacent wells up to an entire column wells (not shown)via counter electrode connections 3851A and/or 3851B (e.g., counterelectrodes 3851A and/or 3851B may extend along an entire column ofwells). Thus, referring to FIG. 34B, three counter electrode contactlocations (*) can be electrically connected to eight wells viaelectrical connections 3851A and/or 3851B and/or via exposed portionswithin wells (e.g., 3855).

Preferably, working electrode contacts 3820, 3821, 3822 and 3823 andcounter electrode contact(s) 3850 are on the bottom of a multi-wellplate. According to a preferred embodiment, such bottom contacts areelectrically connected to the working electrode 3811, 3812, 3813 and3814 and counter electrode surfaces 3854A, 3854B and 3855 via connectivethrough holes, preferably located at locations such as “x” and “*” andthe working electrode connections and/or counter electrode connectionsare on the same plate surface as the corresponding working and counterelectrode surfaces with well 3810. Alternatively, the working andcounter electrode connections can be on the bottom surface and theconductive through hole located beneath the corresponding working andcounter electrode surfaces. According to another embodiment, the workingelectrode connections and/or counter electrode connections are withinone or more patterned intermediate layers (not shown) located betweenthe contact layer and the electrode surface layer. The intermediatelayer(s) would provide electrically isolated conductive paths from theworking electrode and/or counter electrode contacts to the correspondingworking electrode and counter electrode surfaces. The use of anintermediate layer to provide conductive paths would allow for higherdensity arrays of spots within each well since the electricalconnections would not be limited to a two-dimensional configuration.

Using a plate such as that shown in FIG. 38, the electrical connectorsof the apparatus would contact the plate bottom at the four differentcontact locations (e.g., four steps vs. one step) per column of wellswhile the array of photodiodes remained above that column of wells.Referring to FIG. 34B, for example, the single working electricalcontact locations 3480 (represented by X's) shown in the figure would bereplaced with four spaced working electrical contact locations percolumn of wells (e.g., working electrode contacts 3820, 3821, 3822 and3823 of FIG. 38). Each contact/inducement step would result in a voltagebeing applied to one spot per well. The corresponding photodiode foreach well would measure ECL from that spot. Then the second set ofcontacts would be contacted by the connectors to induce ECL at thesecond spot and the same photodiode would measure ECL from the secondspot, then the third set of contacts and finally the fourth. Throughoutthe scan of the 4 spots of a single column of wells, the bottom contactsconnect to different spots as the column of wells would remain under thearray of photodiodes. The different spots under a single photodiode areinduced to emit ECL sequentially thereby allowing a single photodiode todistinguish and measure luminescence from more then one spot. After thefourth spot is induced to emit ECL and the emitted ECL measured, thearray of photodiodes is then shifted to the next column of 4-spot wellsand the process is repeated for each column.

Thus, the only modifications needed to convert the system adapted forreading single-spot 96-well plates to 4-spot 96-well plates is to modifythe electrical contacts on the plate bottoms and change the measurementscan of the plate from a 12 step scan to a 48 step scan (4 spots×12columns) wherein each step involves contacting the plate bottom 48 timesinstead of 12 times, while the array of photodiodes is shifted withrespect to the plate only 12 times. As a result, the same apparatushaving the same fixed array of light detectors can be employed tomeasure ECL from a different plate format. The same methodology can beemployed to measure a 7-spot 96-well plate (e.g., 7 spots×12 colunms=84step scan; 12 spots×12 columns=144 steps, etc.).

Thus, one preferred embodiment involves forming independentlyaddressable working electrodes (and/or counter electrodes) within asingle “sector” (e.g., column of wells) thereby allowing each spot orwell beneath a given photodiode to be measured sequentially.

According to another embodiment, the same array of light detectors canbe used to measure ECL from a 384-well plate. For example, referring toFIG. 2F, a single linear array of photodiodes adapted for measuring ECLfrom a column of wells of a 96-well plate would cover two columns ofwells (e.g., columns 1 and 2) in a 384-well plate. Thus, a singlephotodiode would be used to measure luminescence from each of wells A1,A2, B1 and B2 (a 384 well plate has rows A-P and columns 1-24) bysequentially applying a voltage to each well and a second photodiodewould (simultaneously with the first photodiode) measure luminescencefrom each of wells C1, C2, D1 and D2 by sequentially applying a voltageto each well, etc. The sequential application of voltage to each wellunder the individual photodiode can be achieved by using differentelectrical contacts for each well (e.g., an array of 16 connectors toprovide voltage to each of the 16 wells beneath the array of eightphotodiodes where the connectors apply a voltage to only one well perphotodiode at a time) or by using modified bottom contacts as describedabove with respect to the 4-spot 96-well plate or by the methodsdescribed further below.

According to another embodiment, alternative assay domains or wellswithin a column are induced to emit electrochemiluminescence andmeasured. One preferred embodiment employs 384-well plates wherein eachcolumn of wells would have alternating counter or working electrodes.For example, referring again to FIG. 2F, the counter (or working)electrode in wells A1, C1, E1, G1, I1, K1, M1, O1 would all be connectedelectrically, and the counter (or working) electrode in wells B1, D1,F1, H1, J1, L1, P1 would be connected to each other electrically. Thecorresponding working (or counter) electrode would preferably be commonto all of these wells. First, the A1 group of wells would be excited byconnecting to that counter electrode and the working electrode. Theplate would then be shifted by half the spacing of the 384 well plate(2.25 mm) such that when the contacts were raised to the plate, theywould connect to the working electrode and the counter electrodeconnected to the B1 bank of wells, but not the A1 group of wells. Thusthe second half of the column would be excited and the light measured bythe 8 photodiodes, completing the read of the column. The remaining 23columns of the plate would be read in the same way. Thus, the platewould be shifted to allow the A2 groups of wells to be measured, andshifted again to allow the B2 group of wells, etc. Preferably, it is theplate that is shifting within the apparatus. Alternatively, theelectrical connectors contacting the plate contacts can shift. Thealternating connection to the counter electrodes can be realized eitherby modifying the screen printing to include the counter electrode on oneside of each well bottom in an alternating fashion, or by modifying thedielectric layer to selectively cover the counter electrode in analternating fashion.

For example, referring to FIG. 2B, if counter electrode 226B is coveredby dielectric in every other well in a row of wells (and counterelectrode 226A is covered by dielectric in the wells of that row wherecounter electrode 226B is not covered), then (step 1) applying voltageto the bottom contact corresponding to working electrode 230 (where theworking electrodes for the column of wells are electrically connected)and to the bottom contact corresponding to counter electrode 226B (wherethe counter electrodes for the column of wells are electricallyconnected, but were only exposed in alternating wells) would only resultin ECL in alternating wells. Then (step 2) the contacts connected toworking electrodes 230 and counter electrode 226A would be contacted toinduce ECL in the other set of alternating wells of the same column.This approach would not require fundamental modification to theinstrument, as it would run both plate types with the same mechanicaldesign.

Preferably, the software controlling the instrument is modified toinduce luminescence and/or read luminescence greater than 12 times(preferably at least 48 times, more preferably at least 84 times andmost preferred at least 120 times) rather than the 12 times required forthe standard 96-well multi-array plate using a single linear array oflight detectors.

According to another embodiment, a similar, but preferable approachcould be implemented for using different plate formats (e.g., 96 and 384well plates) with relatively simple electronic modifications to theinstrument. In this case, a switch may be added to the wiring thatapplies voltage to the contacts in the instrument (i.e., to the contactsor connectors that contact the plate bottom). In the unmodifiedapparatus described above, the voltage sweep is applied to redundantcontacts (e.g., see FIG. 34B wherein multiple redundant connectorscontact redundant contact surfaces on the plate bottom for a singlecolumn of wells of a 96-well plate). Thus, without repositioning thecontacts, but instead selectively applying voltages to a subset of thecontacts, it is possible independently addressable plate contacts tofirst excite the A1, C1, etc group of wells centered under thephotodiodes. Then, using appropriate software control, the voltage maybe switched such that it was applied across a different set of contactpins, which would contact other separately addressable plate contacts onthe back of the plate connected to the B1, D1, etc. group of wells,without requiring any motion of the plate.

As another example, as described above, these 4 different electrodescould be addressable by indexing the plate in small steps such that thesame contact points connect to different working electrode leads on thebottom of the plate. Preferably, a switching mechanism is integratedinto the apparatus electronics that provide the connection to theexisting contact mechanism, such that the plate would remain completelystationary and the four spots would be fired in sequence. According toanother embodiment, the electrical mechanisms to provide switching arefield upgradeable or are provided in a different version of theinstrument. The plates and instrument are preferably designed such thatthe standard 96 well plates could still be run on the instrument. Thesoftware controlling the instrument is preferably modified toaccommodate the changes in ECL excitation sequence required and allowthe user to specify what plate type is being used. The contact heightand plate top would preferably remain exactly the same in this case.

The multi-spot or multi-well embodiments described above can begeneralized beyond the specific example of 4 spots to any number ofspots or wells, where the number of addressable spots is limited by theprinting resolution of the screen-printing used to fabricate theindependently addressable electrodes. Alternative manufacturingtechniques (microfabrication and lithography) could be employed toincrease the number of addressable spots beyond what is possible inscreen-printing.

Using different plate formats in the same apparatus may require otherminor adjustments. For example, different plates may have differentplate heights (e.g., the 384 well plate may be shorter than the 96 wellformat). Preferably, a spacer would be used to elevate the shorter384-well plate to the photodiodes and/or the contact height would beadjusted to the correct position (if the instrument is adapted for96-well plates). Alternatively, instead of elevating the height of thecontacts and including a spacer, a full height plate top could be usedwith the 384-well format. This plate top would have the same height asthe 96 well plate top (preferably, the volumes of wells of the 384 aremaintained by raising the well bottom to compensate for the elevatedheight of the plate walls).

Using these embodiments, the excited regions may be somewhat off-centerrelative to the photodiode which may compromise the light collectionand/or cross talk between photodiodes, as compared to measurements usingthe standard 96 well format. This is preferably compensated for by theuser and/or by the software of the system. Alternatively, the array oflight detectors and/or plate may be shifted with respect to each otherto align the well to be induced with the corresponding photodiode. Forexample, the instrument may preferably be further modified to includethe ability to offset the position of the plate (e.g., 384 well plate)in the orthogonal direction, for example, to the standard translation inthe non-modified reader (i.e., along the columns of wells) to centereach well under its photodiode perfectly to optimize the lightcollection and cross talk between sectors.

A still further embodiment of the invention relates to the use of theabove-described approaches in an apparatus having an imaging devicerather than an array of light detectors. For example, sequentiallyfiring spots or wells under an imager could provide for a higher densityof spots or wells for a given level of image resolution. If an imagercannot differentiate light emitted from a group of four tightly packedspots, sequentially firing the spots would allow the user todifferentiate between the spots. Thus, the above-identified approachescan also be applied to an apparatus having an imaging system instead ofan array of light detectors.

According to yet another embodiment, the apparatus having an array oflight detectors could be modified to include an array of 16 photodiodesmatched to the size and spacing of the 384-well plate such that all 16wells in each column could be excited simultaneously. This approachwould have the advantage of reducing the read time compared to the otherapproaches by a factor of 2. In this case, the plate bottom would bedesigned in the same concept as the non-modified 96-well plate, with noalternation between wells in the application of voltages. Thus, anotherembodiment of the invention relates to an apparatus having an array oflight detectors wherein the number of light detectors in the arraycorresponds to the number of wells and/or spots in the column or rowbeing measured.

5.4.3 Assays Methods

The assay plates and instrumentation of the invention are useful forcarrying out a wide variety of established assay formats, e.g., assaysbased on the measurement of photoluminescence, Scintillation ProximityAssay (SPA), chemiluminescence, measurement of electrochemical voltageand/or current or, preferably, an electrode-induced luminescence, mostpreferably, electrochemiluminescence. For examples of methods forconducting ECL assays, the reader is directed towards U.S. Pat. Nos.5,591,581; 5,641,623; 5,643,713; 5,705,402; 6,066,448; 6,165,708;6,207,369; and 6,214,552 and Published PCT Applications WO87/06706 andWO98/12539, these references hereby incorporated by reference. Assaysmay be directed to, but are not limited to, the measurement of thequantity of an analyte; the measurement of a property of a sample (e.g.,temperature, luminescence, electrochemical activity, color, turbidity,etc.); the measurement of a chemical, biochemical and/or biologicalactivity (e.g., an enzymatic activity); the measurement of a kinetic orthermodynamic parameter (e.g., the rate or equilibrium constant for areaction), etc.

The embodiments of the invention can be used to test a variety ofsamples which may contain an analyte or activity of interest. Suchsamples may be in solid, emulsion, suspension, liquid, or gas form. Theymay be, but are not limited to, samples containing or derived from, forexample, cells (live or dead) and cell-derived products, cell fragments,cell fractions, cell lysates, unfractionated cell lysates, organelles,organs, animal parts, animal by-products, cell membranes, cell culturesupernatants (including supernatants from antibody producing organismssuch as hybridomas), immortalized cells, waste or drinking water, food,beverages, pharmaceutical compositions, blood, serum, plasma, hair,sweat, urine, feces, tissue, biopsies, structural biological components,skeletal components (e.g., bone, ligaments, tendons), separated and/orfractionated skeletal components, hair, fur, feathers, hair fractionsand/or separations, skin, skin fractions, dermis, endodermis, effluent,separated and/or fractionated samples, unfractionated samples, separatedand/or fractionated liquids, saliva, mucous, oils, plants, plant parts,plant by-products, sewage, environmental samples, dust, swipes,absorbent materials, gels, organic solvents, chemicals, chemicalsolutions, soil, minerals, mineral deposits, water supply, watersources, filtered residue from fluids (gas and/or liquids), solids,gases, or air. The sample may further comprise, for example, water,organic solvents (e.g., acetonitrile, dimethyl sulfoxide, dimethylformamide, n-methyl-pyrrolidone or alcohols) or mixtures thereof.Analytes and/or other samples that may be measured include, but are notlimited to, whole cells, cell surface antigens, cell nucleus/nuclei,nuclear fractions, subcellular particles (e.g., organelles or membranefragments), membranes, solubilized membranes, membranes fractions,nuclear membranes, nuclear membrane fractions, lipids, lipids withproteins, lipids with sugars, lipid bilayers, micelles, septa,monolayers, separating materials, barriers, dialysis membranes,permeable membranes, nonpermeable membranes, cell membranes, organellemembranes, viruses, prions, eukaryotic cells, prokaryotic cells,immunological cells, fungus, yeast, dust mites or fragments thereof,viroids, antibodies, antibody fragments, antigens, haptens, fatty acids,nucleic acids (and synthetic analogs), proteins (and synthetic analogs),lipoproteins, cytoskeleton, protein complexes, polysaccharides,inhibitors, cofactors, haptens, cell receptors, receptor ligands,lipopolysaccharides, glycoproteins, peptides, polypeptides, cAMP, EGF,kinases, enzymes, enzyme substrates, enzyme products, second messengers,cell signaling factors and/or components, second messenger signalingfactors and/or components, cellular metabolites, hormones, endocrinefactors, paracrine factors, autocrine factors, immunological factors,cytokines, pharmacological agents, drugs, therapeutic drugs, syntheticorganic molecules, organometallic molecules, tranquilizers,barbiturates, alkaloids, steroids, vitamins, amino acids, sugars,lectins, recombinant or derived proteins, biotin, avidin, streptavidin,or inorganic molecules present in the sample.

Activities that may be measured include, but are not limited to, theactivities of phosphorylases, phosphatases, esterases,trans-glutaminases, nucleic acid damaging activities, transferases,oxidases, reductases, dehydrogenases, glycosidases, ribosomes, proteinprocessing enzymes (e.g., proteases, kinases, protein phophatases,ubiquitin-protein ligases, etc.), nucleic acid processing enzymes (e.g.,polymerases, nucleases, integrases, ligases, helicases, telomerases,etc.), cellular receptor activation, second messenger system activation,etc.

In one embodiment of the invention, a sample potentially containing aluminescent, chemiluminescent and/or redox-active substance (preferablyan ECL-active substance) is introduced to an assay plate or one or morewells of an assay plate of the invention and an electrochemical orluminescent signal (preferably, electrochemiluminescence) from thesample is induced and measured so as to measure the quantity of thesubstance. In another embodiment of the invention, a sample containing aluminescent, chemiluminescent and/or redox-active substance (preferablyan ECL-active substance) is introduced to an assay plate or one or morewells of an assay plate of the invention and an electrochemical orluminescent signal (preferably, electrochemiluminescence) from thesample is induced and measured so as to measure the presence ofsubstances, chemical activities or biological activities that affect theproduction of the signal from the substance (e.g., the presence,production and/or consumption of ECL coreactants, hydrogen ions,luminescence quenchers, chemiluminescence triggers, etc.). In otherembodiments of the invention, luminescent, chemiluminescent and/orredox-active substances (preferably an ECL-active substances) are usedas labels to allow the monitoring of assay reagents such as enzymesubstrates or binding reagents. Assays formats for measuring analytesthrough the use of labeled binding reagents specific for the analyteinclude homogeneous and heterogeneous methods.

Preferred assay formats employ solid-phase supports so as to couple themeasurement of an analyte or activity to the separation of labeledreagents into solution-phase and solid phase supported portions.Examples include solid-phase binding assays that measure the formationof a complex of a material and its specific binding partner (one of thepair being immobilized, or capable of being immobilized, on the solidphase support), the formation of sandwich complexes (including a capturereagent that is immobilized, or capable of being immobilized, on thesolid phase support), the competition of two competitors for a bindingpartner (the binding partner or one of the competitors beingimmobilized, or capable of being immobilized, on the solid phasesupport), the enzymatic or chemical cleavage of a label (or labeledmaterial) from a reagent that is immobilized, or capable of beingimmobilized on a solid phase support and the enzymatic or chemicalattachment of a label (or labeled material) to a reagent that isimmobilized or capable of being immobilized on a solid-phase support.The term “capable of being immobilized” is used herein to refer toreagents that may participate in reactions in solution and subsequentlybe captured on a solid phase during or prior to the detection step. Forexample, the reagent may be captured using a specific binding partner ofthe reagent that is immobilized on the solid phase. Alternatively, thereagent is linked to a capture moiety and a specific binding partner ofthe capture moiety is immobilized on the solid phase. Examples of usefulcapture moiety-binding partner pairs include biotin-streptavidin (oravidin), antibody-hapten, receptor-ligand, nucleic acid—complementarynucleic acid, etc.

In assays carried out on solid-phase supports, the amount of analyte oractivity is, preferably, determined by measuring the amount of label onthe solid phase support and/or in solution, measurements typically beingconducted via a surface selective technique, a solution selectivetechnique, or after separation of the two phases. Most preferably, thesolid phase support in the embodiments described above is a workingelectrode of an assay plate or within a well of an assay plate of theinvention; this arrangement allows for surface selectiveelectrochemiluminescent excitation and measurement of labels on thesolid phase support. Alternatively, the solid phase support may be asurface sufficiently distant from a working electrode so that theworking electrode only measures labels in the solution phase.

In assays carried out on solid-phase supports, the amount of analyte oractivity is, preferably, determined by measuring the amount of label onthe solid phase support and/or in solution, measurements typically beingconducted via a surface selective technique, a solution selectivetechnique, or after separation of the two phases. More preferably, thesolid phase support in the embodiments described above is a workingelectrode of an assay plate or within a well of an assay plate of theinvention; this arrangement allows for surface selective excitation ofelectrode-induced luminescence (most preferablyelectrochemiluminescence) and measurement of labels on the solid phasesupport. Alternatively, the solid phase support may be a surfacesufficiently distant from a working electrode so that the workingelectrode only induces luminescence from labels in the solution phase.

In one embodiment of the invention, a reagent labeled with aluminescent, chemiluminescent and/or redox-active label (preferably anECL label) is measured by a method comprising the steps of i)introducing the sample to an assay plate or one or more wells of anassay plate of the invention; ii) contacting the labeled reagent with abinding reagent; ii) forming a binding complex comprising the bindingreagent and the labeled reagent; iii) inducing the labeled reagent toproduce an electrochemical or luminescent signal (preferably,electrochemiluminescence) and iv) measuring the signal so as to measurethe labeled reagent. Preferably, the binding reagent is immobilized orcapable of being immobilized on a solid phase support, the solid phasesupport, most preferably being a working electrode in an assay plate ora well of an assay plate of the invention. The method may also comprisethe step of immobilizing the binding reagent on the solid phase supportand/or working electrode.

The present invention also relates to methods of measuring an analyte ina sample comprising the steps of i) contacting the sample with a labeleddetection reagent and optionally a capture reagent, the detection andbinding reagents having specific binding affinity for the analyte; ii)forming a binding complex comprising the binding reagent, the analyteand, optionally, the capture reagent; iii) inducing the labeleddetection reagent to produce an electrochemical or luminescent signal(preferably, electrochemiluminescence) and iv) measuring the signal soas to measure the analyte in the sample. Preferably, the capture reagentis immobilized or capable of being immobilized on a solid phase support,the solid phase support, most preferably, being a working electrode inan assay plate or a well of an assay plate of the invention. The methodmay also comprise the step of immobilizing the capture reagent on thesolid phase support and/or working electrode.

The present invention also relates to methods of measuring an analyte ina sample comprising the steps of i) contacting the sample with an analogof the analyte and a binding reagent, one of said analog and saidbinding reagent having a label, wherein said analyte and said analogcompete for binding to said binding reagent; ii) inducing said label toproduce an electrochemical or luminescent signal (preferably,electrochemiluminescence) and iii) measuring the signal so as to measurethe analyte in the sample. Preferably, the binding reagent (if theanalog of the analyte has the label) or the analog of the analyte (ifthe binding reagent has the label) is immobilized or capable of beingimmobilized on a solid phase support, the solid phase support, mostpreferably, being a working electrode in an assay plate or a well of anassay plate of the invention. The method may also comprise the step ofimmobilizing the detection reagent or the analog of the analyte on thesolid phase support and/or working electrode.

Another aspect of the invention relates to methods and systems forperforming chemiluminescence assays wherein a chemiluminescent label isinduced to emit luminescence by introducing a trigger, which reacts withthe label to form chemiluminescence. See, U.S. Pat. No. 5,798,083 toMassey et al., hereby incorporated by reference. Preferably, the trigger(such as hydrogen peroxide) is generated by application ofelectrochemical energy at the working electrode. See, U.S. Pat. No.5,770,459 to Massey et al., hereby incorporated by reference. Thegeneration of the trigger by the application of electrochemical energyallows for the timed and/or sequential inducement of chemiluminescencein, for example, the sectors or wells of the assay module.

When chemiluminescence measurements are performed, Applicants have foundit advantageous to adapt the way the background luminescence issubtracted (e.g., how the instrument subtracts a background image).Typically, when performing an ECL measurement, a background image withthe plate positioned under the camera or light detector is taken priorto applying any voltage and the resulting background image is thensubtracted from the image taken while the ECL stimulating voltage isapplied. For the chemiluminescence measurements, this approach can bedisadvantageous if there is chemiluminescence being emitted from thewells during the background read time. However, several differentapproaches can be used to overcome this problem. According to oneembodiment, the apparatus is adapted to take the background image ormeasure the background luminescence before the plate is brought insidethe light tight enclosure (e.g., take an image of the interior of thelight tight enclosure to determine the level of background prior tointroducing the plate into the enclosure). According to anotherembodiment, the apparatus is adapted to take a background image afterintroduction of the plate while the plate is inside the light tightenclosure, but far from the imaged region (i.e., not under the lens orlight detector) so that any chemiluminescence emitted from the platedoes not interfere with the background measurement. According to yetanother embodiment, the apparatus is adapted to characterize thebackground of a given instrument and subtract those values from theprocessed chemiluminescence data, rather than directly subtracting abackground image before processing for each chemiluminescencemeasurement (e.g., provide an “estimated background” for a giveninstrument and use that value for each chemiluminescence measurement).

Yet another aspect of the invention relates to methods for determiningthe rate of a reaction or the time course of reaction using the assaymodules or devices of the invention. See, U.S. Pat. No. 5,527,710 toNacamulli et al. issued Jun. 18, 1996, hereby incorporated by reference.

Surprisingly, after an assay electrode is used in an ECL assay whereinthe electrode is exposed to electrochemical energy to generate ECL, theability of the electrode to induce ECL in a subsequent assay is reduced,but not eliminated. Particularly, if the voltage is kept at a minimum(e.g., close to the minimum required to induce ECL) and/or the durationof time the voltage is applied to induce ECL is minimized, any damage tothe electrodes is minimized or eliminated thereby allowing theelectrodes to be used multiple times. One embodiment of using theelectrodes more than once relates to a method for determining the timecourse of a reaction in which at least one reactant is converted to oneor more products, the method comprising:

-   -   (a) forming a composition containing the reactant and a        luminescent label, wherein        -   (i) the reactant reacts to form a reaction product;        -   (ii) the luminescent label is capable of being induced to            emit a luminescence signal, wherein the luminescence signal            emitted by the luminescent label is affected by the            reaction; and        -   (iii) the luminescence signal emitted changes as the            reaction progresses; and    -   (b) detecting emitted luminescence, preferably at selected time        intervals, to determine the time course of the reaction.

Preferably, a component of the complex (e.g., the reactant or a secondreaction partner) is immobilized on an electrode so that said complex isformed on the electrode. Preferably, the method further comprisesexposing the composition to electrical energy at selected time intervalsand/or measuring the luminescence signal during the selected timeintervals to determine the time course of the reaction.

Preferably, the label is an electrochemiluminescent label.

Preferably, the method further comprises calculating the time course ofthe reaction from the luminescent signals detected in step (b).

Another embodiment of the invention relates to a method for determiningthe time course of a binding reaction comprising:

-   -   (a) forming a composition containing a reactant, a reaction        partner and a luminescent label, wherein:        -   (i) the reactant and the reaction partner bind to form a            complex;        -   (ii) the luminescent label is capable of being induced to            emit a luminescence signal; and        -   (iii) the luminescent label is attached to the reaction            partner, and    -   (b) detecting emitted luminescence, preferably at selected time        intervals, to determine the time course of the reaction.

Preferably, the method further comprises exposing the composition toelectrical energy at selected time intervals and/or measuring theluminescence signal at the selected time intervals to determine the timecourse of the binding reaction.

Preferably, the reaction partner is an antibody and the reactant is anantigen.

According to one preferred embodiment, the reaction partner is attachedto the luminescent label via a covalent bond or via abiotin-streptavidin binding interaction.

Another embodiment relates to a method for determining the time courseof an enzymatic reaction comprising:

-   -   (a) forming a composition containing an enzyme, an enzyme        substrate and a luminescent label, wherein:        -   (i) the enzyme catalyzes the reaction of the substrate to            form a reaction product;        -   (ii) the luminescent label is capable of being induced to            emit a luminescence signal and the luminescence signal            emitted from the luminescent label varies with the            concentration of the substrate or the reaction product; and        -   (iii) the intensity of the luminescence signal emitted            changes as the reaction progresses; and    -   (b) detecting emitted luminescence, preferably at selected time        intervals, to determine the time course of the reaction.

Preferably, the method further comprises exposing the composition toelectrical energy at selected time intervals and/or measuring theluminescence signal at the selected intervals to determine the timecourse of the reaction.

Preferably, the enzyme substrate is a cofactor, more preferably NADH.

Preferably, the reaction product is a cofactor, more preferably NADH.

Another preferred embodiment relates to a method for determining thetime course of a reaction in a composition containing a luminescentlabel wherein the composition is exposed to electrical energy atselected time intervals during said reaction to induce the label to emitan electrochemiluminescent signal and the electrochemiluminescent signalis measured during said selected time intervals to determine the timecourse of reaction.

According to one preferred embodiment, the reaction is a reaction of areactant with a reaction partner to form a reaction product. Preferably,the intensity of the luminescence signal relates to the concentration ofthe reactant, the reaction partner or the reaction product.

Preferably, the reaction is a specific binding reaction of a reactantwith the reaction partner.

Preferably, the reaction is an enzyme catalyzed reaction.

Preferably, the reaction is of a reactant to form a reaction product andthe concentration of said reactant affects said electrochemiluminescentprocess.

A wide variety of materials have been shown to emit electrode inducedluminescence, particularly electrochemiluminescence, and may be usedwith the methods, plates, kits, systems and instruments of theinvention. In preferred electrochemiluminescence systems, ECL-activematerials and/or labels are regenerated after the emission ofelectrochemiluminescence and, during an electrochemiluminescenceexperiment, may be repeatedly excited to an excited state and/or inducedto emit luminescence. For example, one class of ECL-active materials arebelieved to function via a mechanism that includes the steps of i)oxidation of the material; ii) reduction of the oxidized material by astrong reducing agent so as to produce the material in an excited stateand iii) emission of a photon from the excited state so as to regeneratethe ECL-active material. Preferably, the difference in redox potentialsbetween the ECL-active material and the strong reducing agent issufficient so that the energy released by step (ii) is equal to orgreater than the energy of the photon. In an analogous mechanism, steps(i) and (ii) may be replaced by i) reduction of the material and ii)oxidation of the reduced material by a strong oxidizing agent. In someespecially preferred systems, the mechanism is believed to furthercomprises the step of oxidizing an ECL coreactant so as to form thestrong reducing agent or, analogously, reducing an ECL coreactant toform the strong oxidizing agent.

Preferred luminescent materials and labels include luminescentorganometallic complexes of Ru, Os and Re. Some especially usefulmaterials are polypyridyl complexes of ruthenium and osmium, inparticular, complexes having the structure ML¹L²L³ where M is rutheniumor osmium, and L¹, L² and L³ each are bipyridine, phenanthroline,substituted bipyridine and/or substituted phenanthroline. We have foundthat the inclusion of substituted bipyridines or phenanthrolinespresenting substituents comprising negatively charged groups, preferablysulfate groups and most preferably sulfonate groups (as described incopending U.S. patent application Ser. No. 09/896,974, entitled “ECLLabels Having Improved Non-Specific Binding Properties, Methods of Usingand Kits Containing the Same” filed on Jun. 29, 2001, the disclosurehereby incorporated by reference) are especially preferred due to theirresistance to non-specific binding, in particular to electrodescomprising carbon, carbon particles, carbon fibrils, carbon composites,carbon fibril composites and/or carbon inks.

Yet another aspect of the invention relates to methods of reusing theassay modules of the invention. More specifically, a method of using anassay module a second (or third, etc.) time wherein any decrease insignal (e.g., ECL) emitted by the previously used module is compensatedand/or calibrated for in determining the presence or amount of analyteof interest. For example, surprisingly, after an assay electrode is usedin an electrochemiluminescence assay wherein the electrode is exposed toelectrochemical energy to generate ECL, the ability of the electrode toinduce ECL in a subsequent assay is reduced, but not eliminated.Accordingly, one embodiment relates to using an assay module to performa first assay (preferably an electrochemiluminescence assay) and thenusing the assay module to perform a second assay (preferably anelectrochemiluminescence assay), wherein any decrease in signalgenerated by the used assay module is compensated and/or calibrated forin performing the second assay. According to one embodiment, the secondassay described above is a second reading (preferably anelectrochemiluminescence assay) of the same assay in order to generatebetter statistical analysis of the results or to confirm the initialassay determination.

Yet another aspect of the invention relates to methods of refurbishingand/or reconstructing the assay modules after a first use. Morespecifically, methods of reconstituting the electrode surface withbinding reagents to enable the performance of subsequent assays. Oneembodiment comprises removing the used biological reagents from theelectrode surface (e.g., cleaning the electrode surface) and reapplyingbiological reagents to the electrode surface. Another embodiment relatesto reapplying a refurbishing layer (e.g., a carbon layer on a carbonelectrode) over the used biological reagents and then applying newbiological reagents to the refurbishing layer.

The step of removing the used biological reagents can be performed by avariety of methods including washing with solutions such as (i) water,(ii) bleach, (iii) water with surfactant/detergent, (iv) acid solutions,(v) base solutions, (vi) organic solvents (e.g., alcohol, ethanol,methanol, DMSO, acetone, etc.) where the solvent is preferably chosennot to dissolve the material used in the module (e.g., carbon/polymerink electrodes and polystyrene or polypropylene plate) but instead todenature biological materials on the electrode surface, (vii) hydrogenperoxide, (viii) reducing agents (e.g., chemical reduction) on thecarbon surface, (ix) chemical cleaning reaction that will “etch” organicmaterial, (x) electrochemical reduction of cleaned/washed carbonsurface, (xi) electrochemically active solution where applying a voltageto the electrodes during washing will cause the “cleaning”action—preferably including the step of monitoring the electricalproperties (current/voltage) to determine the effect of cleaning, (xii)using elevated temperature solutions to speed the washing, (xiii)multiple washes (e.g., wash 3 times with 200 ul per well to achievebetter cleaning than a single wash—where the number of wash cyclesvaries between 2-10 and the volume varies from 25 to 350 ul (e.g., usingstandard microplate washing protocols and equipment)), and (xiv)combinations of any of these approaches.

The step of removing the used biological reagent and/or otherwiserefurbishing the surface can also be performed using non-liquid/solutionapproaches such as (i) plasma etching, (ii) plasma deposition ofmaterial, (iii) corona treatment, (iv) exposure to ozone, (v) ion orelectron bombardment, (vi) irradiating the carbon surface, (vii) flametreatment of surface, (viii) baking the surface (to drive off material),(ix) baking the surface at reduced pressure, (x) annealing the carbonsurface to reform/refurbish the electrode surface, (xi) combination ofany solution wash as described above with subsequent nonsolutionprocessing, and/or (xii) physical/mechanical treatment (e.g.,sanding/polishing/rubbing, etc.).

Preferably, the refurbished assay module is tested to determine whetherthe refurbishing steps have been sufficient. For example, the electricalproperties (current/voltage) of the refurbished electrode is tested ormonitored to determine the effect of cleaning. Testing can also beperformed visually using an optical microscope or using electronmicroscopy.

The step of coating the washed or otherwise refurbished electrodesurface (preferably carbon electrode surface) with capture reagent canbe performed by (i) microdeposition with or without drying of thedeposited reagent, (ii) coating biological molecules from solution,(iii) using any of the coating approaches described above after theelectrode has already been coated and used once, and/or (iv)electrochemical/chemical/physical/mechanical or any other deposition ofany conductive material onto the surface.

5.5 Systems

Another aspect of the invention relates to a system for conducting aluminescence assay, preferably an electrode induced luminescence assay,more preferably a electrochemiluminescence assay, comprising anapparatus, preferably as described above, for inducing and measuringluminescence and a multi-well plate containing an electrode inducedluminescence reagent, preferably an electrochemiluminescence reagent.

Another embodiment relates to a system comprising the apparatus, amulti-well plate and an electrode induced luminescence reagent,preferably an electrochemiluminescence reagent.

Yet another embodiment relates to a system comprising the apparatus asdescribed above for measuring luminescence and an assay plate,preferably a multi-well assay plate.

Yet another embodiment relates to a system comprising the apparatus forinducing and measuring luminescence and an assay plate, preferably amulti-well assay plate as described above.

A still further embodiment comprises an apparatus and an assay plate,preferably a multi-well assay plate, containing an electrode inducedluminescence reagent, preferably an electrochemiluminescence label.

A still further embodiment comprises the apparatus and one or morerobotic devices and/or systems configured to performing one or more ofthe following functions: (a) moving the plates into, within and out ofthe apparatus, (b) storing the plates (e.g., refrigeration unit), (c)liquid or reagent handling device (e.g., adapted to mix reagents and/orintroduce reagents into wells), (d) assay plate shaker (e.g., for mixingreagents and/or for increasing reaction rates), (e) plate washer (e.g.,for washing plates and/or performing assay wash steps (e.g., wellaspirator)). Such robotic devices and/or systems may be integrated intothe apparatus and/or linked as separate components.

According to a preferred embodiment, the apparatus or systemincorporates (or adjoined to or adjacent to or robotically linked orcoupled to), for example, one or more of the fbllowing devices: platesealer (e.g., Zymark), plate washer (e.g., TECAN, BioTek), reagentdispensor and/or automated pipetting station and/or liquid handlingstation (e.g., Zymark, Labsystems, Beckman, TECAN), incubator (e.g.,Zymark), plate shaker (e.g., Zymark), compound library or sample storageand/or compound and/or sample retrieval module.

According to a preferred embodiment, one or more of these devices arecoupled to the apparatus of the invention via a robotic assembly suchthat the entire assay process can be performed automatically. Accordingto an alternate embodiment, multi-well plates are manually moved betweenthe apparatus and various devices by manually moving stacks of plates.

A particularly preferred embodiment relates to the integration of theapparatus of the invention to a high-throughput assembly. Preferably,the high-throughput assembly comprises one or more of the followingdevices, preferably in series (either by placement or by coupling with arobotic assembly): compound library storage, reagent dispensor and/orautomated pippetting station and/or liquid handling station, incubationand/or shaker station, washer (optional), and the apparatus of theinvention. The system may also comprise a waste disposal module fordisposal of the assay module after the assay is performed.

5.6 Kits

Another aspect of the invention relates to kits for use in conductingassays, preferably luminescence assays, more preferably electrodeinduced luminescence assays, and most preferablyelectrochemiluminescence assays, comprising an assay module, preferablyan assay plate, more preferably a multi-well assay plate, and at leastone assay component selected from the group consisting of bindingreagents, enzymes, enzyme substrates and other reagents useful incarrying out an assay. Examples include, but are not limited to, wholecells, cell surface antigens, subcellular particles (e.g., organelles ormembrane fragments), viruses, prions, dust mites or fragments thereof,viroids, antibodies, antigens, haptens, fatty acids, nucleic acids (andsynthetic analogs), proteins (and synthetic analogs), lipoproteins,polysaccharides, lipopolysaccharides, glycoproteins, peptides,polypeptides, enzymes (e.g., phosphorylases, phosphatases, esterases,trans-glutaminases, transferases, oxidases, reductases, dehydrogenases,glycosidases, protein processing enzymes (e.g., proteases, kinases,protein phophatases, ubiquitin-protein ligases, etc.), nucleic acidprocessing enzymes (e.g., polymerases, nucleases, integrases, ligases,helicases, telomerases, etc.)), enzyme substrates (e.g., substrates ofthe enzymes listed above), second messengers, cellular metabolites,hormones, pharmacological agents, tranquilizers, barbiturates,alkaloids, steroids, vitamins, amino acids, sugars, lectins, recombinantor derived proteins, biotin, avidin, streptavidin, luminescent labels(preferably electrochemiluminescent labels), electrochemiluminescencecoreactants, pH buffers, blocking agents, preservatives, stabilizingagents, detergents, dessicants, hygroscopic agents, etc. Such assayreagents may be unlabeled or labeled (preferably with a luminescentlabel, most preferably with an electrochemiluminescent label). Oneembodiment of the invention includes a kit for use in conducting assays,preferably luminescence assays, more preferably electrode inducedluminescence assays, and most preferably electrochemiluminescenceassays, comprising an assay module, preferably an assay plate, morepreferably a multi-well assay plate, and at least one assay componentselected from the group consisting of: (a) at least one luminescentlabel (preferably electrochemiluminescent label); (b) at least oneelectrochemiluminescence coreactant); (c) one or more binding reagents;(d) a pH buffer; (e) one or more blocking reagents; (f) preservatives;(g) stabilizing agents; (h) enzymes; (i) detergents; (j) desiccants and(k) hygroscopic agents.

Preferably, the kit comprises the assay module, preferably an assayplate, and the assay component(s) in one or more, preferably two ormore, more preferably three or more containers.

Preferably, the assay module is a multi-well plate is adapted for use inconducting the electrode induced luminescence assays (preferablyelectrochemiluminescence assays) in sectors.

According to one embodiment, the kit comprises one or more of the assaycomponents in one or more plate wells, preferably in dry form.

According to one embodiment, the assay components are in separatecontainers. According to another embodiment, the kit includes acontainer comprising binding reagents and stabilizing agents. Accordingto another embodiment, the well reagents may include binding reagents,stabilizing agents. Preferably, the kits do not contain any liquids inthe wells.

One preferred embodiment relates to a kit for use in conductingelectrode induced luminescence assays (preferablyelectrochemiluminescence assays) comprising an assay plate, preferably amulti-well assay plate, and at least one assay component selected fromthe group consisting of at least one luminescent label (preferablyelectrochemiluminescent label) and at least one electrochemiluminescencecoreactant).

Another embodiment relates to a kit comprising a multi-well plate and atleast one electrode induced luminescent label (preferablyelectrochemiluminescent label) and/or at least one bioreagent and/or atleast one blocking reagent (e.g., BSA).

According to one preferred embodiment, the kit comprises at least onebioreagent, preferably immobilized on the plate surface selected from:antibodies, fragments of antibodies, proteins, enzymes, enzymesubstrates, inhibitors, cofactors, antigens, haptens, lipoproteins,liposaccharides, cells, sub-cellular components, cell receptors,viruses, nucleic acids, antigens, lipids, glycoproteins, carbohydrates,peptides, amino acids, hormones, protein-binding ligands,pharmacological agents, luminescent labels (preferably ECL labels) orcombinations thereof.

Preferably, the kit includes immobilized reagents comprise proteins,nucleic acids, or combinations thereof.

According to another embodiment, the kit also comprises an assay diluent(e.g., a reagent into which a reagent is diluted for optimum assayperformance).

According to one preferred embodiment, the plurality of wells includesat least two different bioreagents. For example, a well may include twoor more assay domains, wherein two or more assay domains have differentbioreagent

Preferably, the kit comprises at least one electrochemiluminescencecoreactant and/or at least one electrode induced luminescence label(preferably electrochemiluminescent label).

According to another embodiment, the kit is adapted for multiple assays.Preferably, the kit further comprises an additional assay reagent foruse in an additional assay, the additional assay selected from the groupconsisting of a radioactive assays, enzyme assays, chemical colorimetricassays, fluorescence assays, chemiluminescence assays and combinationsthereof.

According to another embodiment, the kit comprises two or more,preferably four or more, more preferably eight or more, more preferably15 or more and most preferably 25 or more assay modules or plates.According to a preferred embodiment, the kit is contained in aresealable bag or container (e.g., zip-lock opening).

Preferably, the bag or container is substantially impermeable to water.According to one preferred embodiment, the bag is a foil, preferably analuminized foil.

The packaging may be translucent, transparent or opaque. Preferably, theplates are packaged in aluminum lined plastic containers or bagscontaining a dry or inert atmosphere (e.g., the bags may be sealed underan atmosphere of nitrogen or argon or the bags may contain a desiccant).According to another embodiment, the containers are vacuum sealed.

Preferably, the container contains 1 plate. According to anotherembodiment, the container contains ten plates. According to anotherembodiment, the container includes between and 100 plates.

Preferably, the assay modules or plates are sterile and/or substantiallyfree of dust and other contaminants.

Preferably, the assay modules are also substantially sterile.

According to one embodiment, the kit is manufactured (at least in part)and/or packaged in a “clean room” environment. Preferably, the kit ismanufactured (at least in part) and/or packaged in a Class 100,000 cleanroom having <100,000 particles (the clean room particle count using a0.5 micron particle count number) per cubic foot (or 3.53 millionparticles per cubic meter).

Preferably, the contaminant particle counts (particles less than 0.5microns) of the kit is less than 60 million per square meter, morepreferably 30 million per square meter, even more preferably less than20 million, even more preferably less than 15 million and mostpreferably less than 10 million.

Preferably, any contaminating non-volatile residue is less than 0.50g/meter², more preferably less than 0.25 g/meter², even more preferablyless than 0.15 g/meter² and most preferably less than 0.10 g/meter².

Preferably the contaminant ion concentration is less than 50 ppm, morepreferably less than 20 ppm, even more preferably less than 10 ppm, evenmore preferably less than 5 ppm, and most preferably less than 1 ppm.

Another aspect of the invention relates to novel approaches forstabilizing various biological/chemical species coated onto assayelectrodes or assay modules or the like, preferably onto the electrodesof the multi-well plates of the invention. For example, multi-wellplates or kits may be manufactured for a range of applications. Theplates may be uncoated or pre-coated with specific reagents. Typically,the reagents coated onto the plate enable specific binding of some assayconstituent to the plate surface. Once these reagents are coated ontothe plates, there will usually be some time delay before the plates areused in an assay. Therefore, the stability of the reagent coating iscritical. The reagent may become less biologically active or becomeinactive if it denatures or otherwise degrades. The approaches ofstabilizing coatings described below can be applied to the differenttypes of coatings and/or different assay modules (e.g., multi-wellmulti-spot plates, etc.) described throughout this specification.

One embodiment for stabilizing reagents on the plate surface involvesapplication of a stabilizing solution to the plate and subsequent dryingof this solution on the surface prior to, during or after theapplication of the biological reagents. Preferably, the stabilizingsolution is a sugar containing, buffered solution. When dried, thesolution leaves a coating of sugar that creates a desirable environment,which promotes stability of the biological activity of the immobilizedreagents. Preferably, the resultant coated surface comprises between 1to 100 μg/cm² of sugar. The amount of sugar present on the surface canbe measured by re-hydrating the wells with an aqueous solution andmeasuring the amount of sugar that dissolves into the solution.

One embodiment employs a stabilizing solution comprising: (a) a buffer(e.g., ammonium phosphate, sodium phosphate, and/or potassium phosphate)and (b) a sugar. The sugar can be any one of the family of simple sugarsincluding fructose, maltose, sucrose, glucose, trehelose, etc.Preferably, the sugar is sucrose.

According to another embodiment, the stabilizing solution furthercomprises a preservative (e.g., Kaython (a commercial preservative)).According to another embodiment, the stabilizing solution furthercomprises a surfactant, preferably a nonionic surfactant (e.g., Tween20). According to yet another embodiment, the stabilizing solutionfurther comprises the preservative and the surfactant. However, thestabilizing solution could optionally comprise the buffer and the sugar,without a preservative or surfactant.

According to a preferred embodiment, the stabilizing solution comprises:from 10 to 30 g/l ammonium dihydrogen-phosphate; 1 to 2 g/l ammoniummonobasic phosphate; 1 to 3 g/l Kaython (commercial preservative); 0.5to 2 g/l Tween 20 (a commercial surfactant); and 10 to 30 g/l sucrose.

According a particularly preferred embodiment, the stabilizing solutioncomprises: 24.7 g/l ammonium dihydrogen-phosphate; 1.5 g/l ammoniummonobasic phosphate; 2 g/l Kaython (commercial preservative); 1 g/lTween 20 (a commercial surfactant); and 20 g/l sucrose.

Preferably, the pH of the solution is adjusted to between 6.5 and 8.5,more preferably between 7.0 and 8.0, even more preferably between 7.4and 7.8 and most preferably about 7.6. Preferably, the pH is adjustedwith either a simple acid or simple base, such as potassium hydroxide(base) or hydrogen chloride (acid).

The invention also relates to methods of applying the stabilizingsolution. There are several ways to apply the stabilizing solution tothe surface (e.g., electrode or plate) according to the invention.Typically, a capture reagent is micro-dispensed onto the working area(s)(e.g., assay spots, assay regions or assay domains) of the workingelectrode(s). After an incubation period, the wells are optionallywashed and/or blocked with a blocking reagent. If the plates are notblocked, the stabilizing solution may be used to wash off unboundcapture reagent, leaving a small amount of stabilizing solution to dryin the well. Typically, only a thin film of stabilizing solution is leftin the well (e.g., quantities less than 5 ul are preferable for eachwell of a 96 well plate while quantities less than 2 ul are preferablefor each well of a 384 well plate). According to another embodiment, theplates are blocked wherein the blocking solution is aspirated from thewells, and the remaining solution is washed away with the stabilizingsolution. A small amount of stabilizing solution is left in the well tocoat the biological reagents wherein both the capture reagents and theblocking reagents are stabilized by the stabilizing solution.

Another embodiment of the invention relates to a method of properlystoring the assay modules of the invention. In order to maintainstability over long storage times, the plates should be dry. Severalsteps may be employed to ensure the dryness of the plates. First, mostof the stabilizing solution is preferably removed at the end of the washcycle. The remaining fluid is typically dried in ambient conditions forsome period of time. Alternatively, the wells may be dried by blowingdry air into the wells for a period of time. According to oneembodiment, the air is blown through individual tubes into each well ofthe plate. Alternatively, air can be sucked through tubes placed in eachwell causing the relatively dry air in the room to flow into the wells.Both approaches achieve similar drying of the wells. Another method ofdrying the plates is to place batches of plates that have been washedwith stabilizing solution into a vacuum chamber. The remaining waterevaporates in the vacuum leaving the plates dry. The drying of thestabilizing solution on the plates can be achieved in individual batchesor as part of an automated plate-processing system.

After the plates are dried with the stabilizing solution, they arepreferably packaged with desiccant packs to ensure that further dryingmay take place. The desiccant may also absorb any detrimental watervapor that penetrates the packaging material over the shelf life of theproduct. Preferably, high barrier materials are used for packaging toprevent the penetration of water vapor. Preferably, the atmosphere inthe package is removed as the plates are vacuum-packed. Prior tovacuum-packaging, the package can be filled with an inert gas (e.g.,nitrogen, argon) to displace the oxygen and water vapor from thepackage. According to one preferred embodiment, the plates are containedin a bag comprising polypropylene laminated to an aluminum foil (e.g.,0.35 mil aluminum foil) and containing a desiccant (e.g., preferablysilica gel, more preferably about 3.5 grams silica gel desiccant witheach packaged plate). The exact quantity of silica gel desiccantrequired depends on the permeability of the packaging material to watervapor, the residual water on the plate, and the desired shelf life ofthe product. In one preferred embodiment, between 1 and 2 grams ordesiccant is used per plate. Preferably, the desiccant will change colorafter absorbing a threshold amount of water. The amount of desiccantrequired increases when multiple plates are packaged together, bothbecause of the increased water remaining on the plates and because ofthe larger surface area of the packaging.

Another embodiment of the invention relates to novel methods ofmeasuring the drying or dryness of an assay module by measuring thechange in conductivity of the surface on which the reagents areimmobilized. For example, the multi-well plates of the invention haveintegrated electrodes on the bottom of each well allowing for themeasurement of the dryness of the plate bottoms in a way that is notpossible with standard multi-well plates. The stabilizing solution thatcoats the well bottom and well walls is a conductive material and theconductivity of the solution depends on the concentration of water.Thus, as the solution forms a coating and dries, the conductivitydecreases until it reaches a steady state. By measuring the conductivityfrom the working to counter electrode, it is possible to monitor thedegree of dryness, serving as a quality control measurement for platedrying. A similar measurement can be performed after storage of thepackaged plates to confirm that the packaging provided a good barrier towater and/or that the desiccant was sufficient to keep the plate dry. Toensure stability of the plates, the plates are dried until theconductivity is less than 30 μS (microsiemens), preferably less than 10μS, more preferably less than 5 μS, and most preferably less than about1 μS.

5.7 Adaptor for Non-Conforming Plate

Another aspect of the invention relates to plate adaptor designed andconfigured for use with an apparatus for conducting electrode inducedluminescence assays (preferably electrochemiluminescence assays) usingplates having contact surfaces not aligned with the electricalconnectors of the apparatus. Thus, an adaptor is employed to allow foran adaptive electrical connection between the electrical connectors ofthe apparatus and the contact surfaces of the non-conforming plate.

One embodiment of the invention relates to a plate contact adapter foruse in an apparatus for conducting assays in an assay plate, preferablymulti-well assay plate having a plurality of wells, comprising aplurality of plate working contact surfaces and a plurality of platecounter contact surfaces for providing electrical energy to theplurality of wells, the apparatus including one or more workingconnectors and one or more counter connectors adapted to provideelectrical energy to the plurality of wells, wherein the one or moreworking connectors and/or one or more counter connectors do not mateand/or are not aligned with the corresponding plate working contactsurfaces and/or plate counter contact surfaces, the plate contactadapter comprising:

(a) a nonconductive substrate having at least a first adaptor surfaceand a second adaptor surface;

(b) one or more working adaptor contact surfaces on the first adaptorsurface configured to mate with the one or more working connectors ofthe apparatus;

(c) one or more counter adaptor contact surfaces on the first adaptorsurface configured to mate with one or more counter connectors of theapparatus;

(d) one or more working adaptor contacts on the second adaptor surfaceelectrically connected to the one or more working adaptor contactsurfaces and configured to come into electrical contact with one or moreof the plate working contact surfaces; and

(e) one or more counter adaptor contacts on the second adaptor surfaceelectrically connected to the one or more counter adaptor contactsurfaces and configured to come into electrical contact with one or moreof the plate counter contact surfaces.

Preferably, the plate adapter has dimensions roughly corresponding to astandard 96-well or 384-well plate and adapter contact surfaces atadapter contact locations that are located at at least one, preferablyat least two, more preferably at least four and most preferably all, ofthe following locations on the first adapter surface, the locationsbeing defined by coordinates (X, Y) measured (inches, ±0.250″,preferably ±0.125″) from the left and top edges, respectively, of theplate adapter

-   -   (i) one or more (preferably two or more, more preferably three        or more and most preferably all) of first sector locations:        (0.743, 0.620), (1.097, 0.620), (1.451, 0.620), (0.743, 1.329),        (1.097, 1.329), (1.451, 1.329), most preferably, one or more        working adapter contact locations selected from (0.743, 0.620),        (1.451, 0.620), (0.743, 1.329) and (1.451, 1.329) and/or one or        more counter adapter contact locations selected from (1.097,        0.620), and (1.097, 1.329);    -   (ii) one or more (preferably two or more, more preferably three        or more and most preferably all) of second sector locations:        (2.161, 0.620), (2.515, 0.620), (2.869, 0.620), (2.161, 1.329),        (2.515, 1.329), (2.869, 1.329), most preferably, one or more        working adapter contact locations selected from (2.161, 0.620),        (2.869, 0.620), (2.161, 1.329), and (2.869, 1.329) and/or one or        more counter adapter contact locations selected from (2.515,        0.620), and (2.515, 1.329);    -   (iii) one or more (preferably two or more, more preferably three        or more and most preferably all) of third sector locations:        (3.579, 0.620), (3.933, 0.620), (4.287, 0.620), (3.579, 1.329),        (3.933, 1.329), (4.287, 1.329), most preferably, one or more        working adapter contact locations selected from (3.579, 0.620),        (4.287, 0.620), (3.579, 1.329), and (4.287, 1.329) and/or one or        more counter adapter contact locations selected from (3.933,        0.620), and (3.933, 1.329);    -   (iv) one or more (preferably two or more, more preferably three        or more and most preferably all) of fourth sector locations:        (0.743, 2.038), (1.097, 2.038), (1.451, 2.038), (0.743, 2.747),        (1.097, 2.747), (1.451, 2.747), most preferably, one or more        working adapter contact locations selected from (0.743, 2.038),        (1.451, 2.038), (0.743, 2.747), and (1.451, 2.747) and/or one or        more counter adapter contact locations selected from (1.097,        2.038), and (1.097, 2.747);    -   (v) one or more (preferably two or more, more preferably three        or more and most preferably all) of fifth sector locations:        (2.161, 2.038), (2.515, 2.038), (2.869, 2.038), (2.161, 2.747),        (2.515, 2.747), (2.869, 2.747), most preferably, one or more        working adapter contact locations selected from (2.161, 2.038),        (2.869, 2.038), (2.161, 2.747), and (2.869, 2.747) and/or one or        more counter adapter contact locations selected from (2.515,        2.038), and (2.515, 2.747); and    -   (vi) one or more (preferably two or more, more preferably three        or more and most preferably all) of sixth sector locations:        (3.579, 2.038), (3.933, 2.038), (4.287, 2.038), (3.579, 2.747),        (3.933, 2.747), (4.287, 2.747), most preferably, one or more        working adapter contact locations selected from (3.579, 2.038),        (4.287, 2.038), (3.579, 2.747), and (4.287, 2.747) and/or one or        more counter adapter contact locations selected from (3.933,        2.038), and (3.933, 2.747).

Preferably, the adapter comprises a first layer comprising one or moreadapter contact surfaces, a second layer comprising one or more adaptercontacts and an insulating layer(s) in between. Preferably, theinsulating layer(s) includes one or more conductive pathwayselectrically connecting the one or more adapter contact surfaces withthe one more adapter contacts. According to one preferred embodiment theadapter comprises an adhesive on the surface having the adaptor contactsso that the adapter can be affixed to a plate bottom. According toanother embodiment, the adaptor clamps onto the plate. According to yetanother embodiment, the adapter is configured to connect to theelectrical connectors of the apparatus.

Another embodiment of the invention relates to a method for measuringluminescence from a multi-well assay plate having a plurality of wellscomprising providing electrical energy to the multi-well assay plateusing an adapter.

Another embodiment relates to an apparatus comprising the adapter.

Yet another embodiment relates to a multi-well plate with an adapteraffixed thereto or in contact therewith.

5.8 Method of Re-Focusing Wells

Another aspect of the invention relates to a method of conducting one ormore assays using an apparatus for measuring luminescence from amulti-well assay plate having a plurality of wells, the apparatuscomprising a source of energy for generating luminescence within theplurality of wells and a camera for measuring luminescence emitted fromthe plurality of wells, the method comprising optimizing the measuringby adjusting the camera focus thereby optimizing the method ofconducting the one or more assays in the apparatus. Preferably, theoptimizing comprises adjusting a lens and/or adjusting the distancebetween the wells and the camera. Such methods, for example, allow forthe use of plates having different dimensions (i.e., the distancesbetween the imaging or detecting surface and the emitting surfacevaries) and/or different volumes of fluid within the wells and/ordifferent sample compositions (e.g., having different opticalproperties).

Preferably, the apparatus is adapted to allow for re-focusing to allowfor the detection of the presence of a lid or cover and the subsequentcompensation for the effect of that lid or cover on the image of theplate bottom. Additionally, the re-focusing would allow the detection ofthe amount of fluid in the well (or a change in the index of refractionof the fluid in the well) and subsequent refocusing on the plate bottom.

5.9 Method of Rearranging the Electrical Contacts or ElectricalConnectors

Another aspect of the invention relates to methods of conducting one ormore assays using multi-well plates having different contact surfaceconfigurations. That is, a method comprising re-configuring orrearranging the electrical connectors of the apparatus to properly alignand contact the contact surfaces of the plate. Such methods allow forgreater flexibility in plate selection. Such methods also allow for thefuture use of future plates having new contact configurations.

One embodiment of the invention relates to a method of conducting one ormore assays using an apparatus for measuring luminescence from amulti-well assay plate having a plurality of wells and two or more platecontact surfaces electrically connected to the plurality of wells, theapparatus comprising a source of energy for generating luminescencewithin the plurality of wells, a camera for measuring luminescenceemitted from the plurality of wells and one or more electricalconnectors for contacting the multi-well plate thereby providing energyto the wells, the method comprising arranging and/or reconfiguring theone or more electrical connectors of the apparatus to align with theplate contact surfaces.

Another embodiment of the invention relates to a method where a firstset of electrical connectors is replaced with a second set having adifferent configuration. Thus, another aspect of the invention relatesto an apparatus adapted to provide for such re-configuration orre-positioning of the electrical contacts and/or replacement of a firstset of electrical contacts with a second set having a differentconfiguration.

5.10 Computer Implemented Control/Interface System

CCD Implementation

FIG. 35a depicts a top level block diagram for one embodiment of anautomated/computerized process for carrying out the ECL-based assays ona representative diagnostic device as described herein. It should beunderstood that any one of, or all of, the steps depicted in the processflow diagram in FIG. 35a and any related figures may be implemented by ageneral purpose computer system or by a specially designed/outfittedcomputer system. A typical computer system would consist of at least oneprocessor and at least one memory coupled to the processor. In oneembodiment, the process flow depicted in FIG. 35a and the relatedfigures may be embodied in a set of instructions that can be executed bya processor. In such an embodiment, the set of instructions forperforming ECL-based assays on an instrument coupled to a computer maybe stored in a computer readable storage medium including, for example,any magnetic medium, any optical medium, any magneto-optical medium, andthe like. The computer readable storage medium may be accessed: in alocal fashion such as, for example, by direct access to read only memory(ROM) or by loading the storage medium containing the executableinstructions into an appropriate reading device locally coupled to thecomputer system; or in a remote fashion such as, for example, bydownloading the set of instructions from a device remotely coupled tothe computer system. A device remotely coupled to the computer systemmay, for example, include a server networked to the computer system, aremote storage device or dedicated network appliance, a digitaltransmission device (e.g., satellite, microwave, infrared and/or radiobroadcast) or the like.

At step 3520 at least a portion of the set of instructions is loadedinto a memory coupled to the processor for execution by the processor.FIG. 35b is a more detailed block diagram illustrating the operationscarried out at step 3520. In one embodiment, initialization of theinstructions may include reading a configuration file 3521 that containsinformation related to the configuration of the instrument such as, forexample, information related to the specific camera, information relatedto the motion control system, information related to the ECLelectronics/subsystem, and the like. Camera specific information mayinclude, for example, camera type (CCD, CMOS), parameters relating tothe specific CCD chip, operating parameters (integration time, binning,gain settings, etc.), defect maps, filters, fixed focal length orvariable focal length, and the like. Information related to the controlsystem may include, for example, number and placement of motors, numberand placement of position sensors, degrees of freedom, ranges of motion,velocity profile parameters, viable paths, the presence or absence of arobotic loading system (e.g., robotic system to load each plate or toload each stack tube, etc.), and the like. Information related to theECL electronics/subsystem may include, for example, power source, rangeof waveforms that can be applied, number and position of electricalcontacts, range of motion of electrical contacts, and the like. Once theconfiguration file is read 3521, the computer system may executeappropriate instructions to initialize the camera and related subsystems3522, initialize the motion controller and related subsystems,initialize the ECL electronics and/or related subsystems and initializeany other systems or subsystems which may be defined in theconfiguration file read at 3521.

At step 3502, the stack tubes are loaded into their respectivereceptacles. Loading of the stack tubes may occur in an entirelyautomated fashion, in an entirely manual fashion, or in any combinationthereof. For example, in an entirely automated fashion, a computersystem coupled to a robotic manipulation system could containinstructions for directing the robotic manipulation system toload/unload the stack tubes. Of course it should be understood that thecomputer system coupled to the robotic manipulation system could be thesame computer system coupled to the diagnostic device or could be aseparate computer system dedicated to controlling the roboticmanipulation system. At step 3530 an instruction is issued to the motioncontrol system to load a plate onto the plate carrier. FIG. 35c is amore detailed block diagram illustrating the operations carried out atstep 3530. At step 3531 the computer system carries out the instructionsdirecting the appropriate subsystems to open the enclosure's door 3531and move the plate carrier into position 3532, 3533 for receiving aplate from the stack tube containing the plates to be analyzed. Once theplate carrier is in position for receiving a plate, the computer carriesout the instructions 3534, 3535 directing the appropriate subsystems torelease a plate from the stack tube onto the plate carrier. At steps3536, 3537 the appropriate subsystems are directed to engage the plateretention mechanism on the stack tube and move the plate carrier intoposition for subsequent transport into the enclosure of the diagnosticdevice. In one embodiment, as depicted in FIG. 35e , mechanical latchesmay be actuated by solenoids to release/retain 3534/3536 the platesfrom/in the stack tube. In a preferred embodiment, sensors wouldindicate whether or not a plate has been successfully loaded onto theplate carrier 3538. If the sensors indicate to the computer system thata plate is present, instructions directing the appropriate subsystems tomove the plate carrier into the enclosure 3545 and close the enclosure'sdoor 3546 are executed. If however, the sensors indicate that a platehas not been loaded onto the plate carrier, alternate instructions areexecuted directing the appropriate subsystems to execute theinstructions for implementing steps 3533 through 3539 once again. Atstep 3539 if it is determined that a plate is still not present, thecomputer first determines whether this operation has been attemptedbefore and if so 3540 executes an alternate set of instructionsdirecting the appropriate subsystems to move the plate carrier back intothe enclosure 3541 and generate a fault 3542, terminating the process3549. Of course it should be understood that FIG. 35c depicts oneillustrative embodiment where an attempt to load a plate onto the platecarrier is made only twice and that the number of attempts made to loada plate could vary from as little as once to as many times as isspecified; the number of attempts could be a fixed number prescribed inthe set of instructions or it could be a variable number eitherprescribed by the configuration file loaded at 3521 or by theuser/operator.

Next, after a plate has been loaded onto the plate carrier at 3530, abar code 3730-3733, 3740-3743 as depicted in FIGS. 37A-37C for oneillustrative embodiment, is read from the plate 3550. FIG. 35d is a moredetailed block diagram illustrating the operations carried out at step3550. At step 3551 the computer system determines whether a firstposition, e.g., 3730 is occupied by bar code information. In oneembodiment, the user/operator can specify the number and placement ofbar codes on the plate, for example, through a user input device such asa mouse, keyboard, data file, and the like. In another embodiment, thefirst position 3730 itself could contain encoded information indicatingthe number and placement of any bar codes. In such an embodiment, thecomputer system could, for example, execute instructions which direct itto always read the first position and use the information from the firstposition to determine the number and placement of other bar codedinformation. If the first position contains bar code information, thecomputer system would instruct the appropriate subsystems to move theplate carrier to the first bar code reading position 3552 and read 3553the bar code information 3730. This process could then repeat 3554-3556for any number of subsequent bar code positions 3730-3733, 3740-3743occupied by bar code information until all bar codes specified have beenread. It is important to note that FIG. 35d illustrates only onepossible embodiment where two bar codes are possible and that any numberof bar codes (see e.g., FIGS. 37A-37C) may in practice be utilizedlimited only by physical constraints (e.g., readable areas of the plate,number of bar codes that can be placed on the readable areas of theplate, etc.).

In a preferred embodiment, the system can use a software design patternknown as a “chain of responsibility” to allow microtiter plates to beprocessed based on bar codes. The diagnostic device may be configured toread and interpret bar codes on microtiter plates. Bar codes may have amultitude of information encoded in them in a multitude of variousformats. The chain of responsibility pattern allows the computer systemto read and interpret many different formats. Some formats may have beenspecified at the time of manufacture or assembly of the diagnosticdevice while others may be specified at some later point in time whilestill others may be specified by various parties.

For instance, in one example the manufacturer of the diagnostic devicemay have specified certain requirements and/or limitations for thenumber and types of assays, the type of microtiter plates, the number ofwells, the number of spots within wells, the number, type, compositionand/or placement of electrodes, and the like, that the particular devicecan handle. In another example the manufacturer of the microtiterplates, or a portion of the microtiter plates (e.g., the microtiterplate without the plate bottom, the plate bottom with integralelectrodes, etc.), which may or may not be the same as the manufacturerof the diagnostic device itself, may also have certainrequirements/limitations that it has specified for proper handling anduse of its plates (e.g., materials used, processes used in fabricatingelectrodes, etc.).

In still another example the party responsible for immobilizing certainreagents on the electrode, which may or may not be the same party aseither the microtiter plate or diagnostic device manufacturer, may havespecified further its own requirements/limitations for proper use andhandling of the microtiter plates it has processed with its reagents(e.g., temperature, moisture, light/UV exposure, shelf life, storagerequirements, which wells contain controls (positive and/or negative),known calibrators with specific concentrations, unknown samples, and thelike). In yet another example, the party performing assays may wish toapply bar codes to the plates in order to track which compounds havedispensed into which wells.

Use of a chain of responsibility approach allows new formats to beintroduced at any time and by any party. A party wishing to place a barcode upon the microtiter plate may do so by simply providing acomponent, a bar code interpreter, which may be added to the systemwithout the need for existing code to be re-written or modified. Eachbar code interpreter could be a self-contained component that implementsa generic interface for parsing and decoding a particular bar codeformat. When a bar code is read, the computer system executesinstructions which assigns the task of identifying an appropriate barcode interpreter to another set of instructions which constructs a listof the available bar code interpreters, and asks the first interpreterto decode the bar code.

In one embodiment, if the first interpreter understands the encoding, itparses and decodes the information on the bar code; if the interpreterdoes not recognize the format, it passes the bar code on to the nextinterpreter on the list. In this way, each interpreter is given a chanceto process the bar code. In another embodiment, the computer systemcould pass the encoded information to each interpreter, either inparallel or in successive fashion, and await a response as to whetherthe interpreter recognizes the format. If a new format is introduced,the system need only be configured with a new interpreter to handle thenew format. Other interpreters in the chain will ignore the new formatand either instruct the computer system that it does not recognize theformat or pass it on to the next interpreter in the chain until theproper interpreter for the format is found.

Once the bar code information has been read at 3550 instructions areexecuted which direct the appropriate subsystems to “read” the plate3560. FIG. 35e is a more detailed block diagram illustrating theoperations carried out at step 3560. At step 3561 instructions areexecuted which direct the appropriate subsystems to move the platecarrier into position to read the first sector. Once in position forreading of the first sector, instructions are executed which direct theappropriate subsystems to read a background image 3562, 3563. At step3564 instructions are executed to perform raw image processing on thebackground image taken at 3563. Step 3564 could include, for example,execution of instructions and application of certain algorithms toperform CCD chip defect correction, transposition (if necessary), cosmicray artifact removal and calculation of image statistics 3576 (imageprocessing is discussed in greater detail below).

After the image taken at 3563 has undergone raw image processing, thebackground image processing step 3565 is performed in order to determinewhether a light leak condition exists 3577. If there is not adetermination of a light leak condition the process continues to step3566 where the appropriate subsystems are directed to raise theelectrical contacts 3566, apply the appropriate waveform 3567 and beginthe camera acquisition procedure 3568. As depicted in FIG. 35e , steps3567 and 3568 are conducted substantially in parallel; i.e., in asubstantially simultaneous manner. After the camera acquisitionprocedure has terminated at 3569 the image acquired is read at 3570 andsubjected to raw image processing 3571, which could include one or moreof CCD chip defect correction, transposition, cosmic ray artifactremoval and calculation of image statistics 3576.

Next, the image processed at 3571 is passed to a sector image processingprocedure 3572. In one embodiment, step 3572 could include one or moreof resizing, background subtraction, creation and application of a wellmask, auto-centering, averaging, cross-talk correction, dark imagedetection and saturation detection. The appropriate subsystems are thendirected to lower the electrical contacts 3573. If there are anyremaining sectors to be read 3574 the appropriate subsystems aredirected to move the plate carrier to the next sector 3575 and thisprocess continues until all of the sectors on the plate have been read3579. Of course it should be understood that steps 3571 and 3572 neednot be conducted in a real time manner but instead may be performed inan off-line mode; e.g., the diagnostic device can read all the sectorson an individual plate, all the plate in a stack tube or any number ofplates, and store the images for subsequent image processing. In thismanner of operation, one embodiment could allow a user to initiate theimage processing by executing an image processing routine or anotherembodiment could allow image processing to be automatically scheduled tooccur at a predetermined time, after a predefined number ofplates/sectors have been read, or any combination thereof.

After the entire plate has been read instructions are executed thatdirect the appropriate subsystems to eject the plate 3580. FIG. 35f is amore detailed block diagram illustrating the operations carried out atstep 3580. In response to directions to eject the plate from theenclosure, the appropriate subsystems open the enclosure's door 3581.Next, it is determined whether or not the plate is to be returned to thein-stack position (e.g., running the device with a robotic manipulationsystem) and if so, the appropriate subsystems are directed to move theplate carrier to the “in-stack” position. If it is determined that theplate is not to be returned to the in-stack position, the appropriatesubsystems are directed to move the plate carrier to the “out-stack”position. In either case, whether positioned at the in-stack position orthe out-stack position, the appropriate subsystems are directed to raisethe plate carrier, or raise the stacker lift, so that the plate isplaced into the appropriate receptacle. The stacker lift is then lowered3586 and in a preferred embodiment a determination is made as to whetheror not the plate has been successfully ejected from the plate carrier3587, 3588. If the plate has not been successfully ejected, steps 3585through 3588 are repeated for as many times as specified or until theplate has been ejected. While FIG. 35f depicts an iterative process forsteps 3585 through 3588 which is performed only twice, it should beunderstood that this process can be carried out for any number ofiterations. Once the iteration count has exceeded the allowable amount3589, the appropriate subsystems are directed to move the plate carrierinto the enclosure 3590 and generate a fault 3591. If the plate has beensuccessfully ejected as determined at step 3588, the appropriatesubsystems would be instructed to move the plate carrier into theenclosure 3592.

Photodiode Array Implementation

FIG. 36a depicts a top level block diagram for one embodiment of anautomated/computerized process for carrying out the ECL-based assays ona representative diagnostic device that utilizes a photodiode array asdescribed herein. It should be understood that any one of, or all of,the steps depicted in the process flow diagram in FIG. 36a and anyrelated figures may be implemented by a general purpose computer systemor by a specially designed/outfitted computer system. A typical computersystem would consist of at least one processor and at least one memorycoupled to the processor. In one embodiment, the process flow depictedin FIG. 36a and the related figures may be embodied in a set ofinstructions that can be executed by a processor. In such an embodiment,the set of instructions for performing ECL-based assays on an instrumentcoupled to a computer may be stored in a computer readable storagemedium including, for example, any magnetic medium, any optical medium,any magneto-optical medium, and the like. The computer readable storagemedium may be accessed: in a local fashion such as, for example, bydirect access of read only memory (ROM) or by loading a storage mediumcontaining the executable instructions into an appropriate readingdevice locally coupled to the computer system; or in a remote fashionsuch as, for example, by downloading the set of instructions from adevice remotely coupled to the computer system. A device remotelycoupled to the computer system may, for example, include a servernetworked to the computer system, a remote storage device or dedicatednetwork appliance, a digital transmission device (e.g., satellite,microwave, infrared and/or radio broadcast) or the like.

At step 3620 the diagnostic device is powered up and at least a portionof the set of executable instructions is loaded into a memory coupled tothe processor for execution by the processor. FIG. 36b is a moredetailed block diagram illustrating the operations carried out at step3620. In one illustrative embodiment, the diagnostic device is initiallypowered up by a user/operator 3621 and the user/operator can elect toload a plate at this time 3622. If the user/operator wishes to load aplate, touching the door sensor 3623 activates the appropriatesubsystems to open the unit's cover 3624. Once the cover has beenopened, the user/operator can load a plate 3625 and close the door 3626by touching the door sensor 3627. Touching the door sensor 3627activates the appropriate subsystems to close the cover 3628. At thispoint the program/software can be started up 3629 by the user/operatorto begin operation of the device.

At step 3640 at least a portion of the set of instructions is loadedinto a memory coupled to the processor for execution by the processor.FIG. 36c is a more detailed block diagram illustrating the operationscarried out at step 3640. In one embodiment, initialization of theinstructions may include reading a configuration file 3641 that containsinformation related to the configuration of the instrument such as, forexample, information related to the specific photodiode sensors,information related to the motion control system, information related tothe ECL electronics/subsystem, and the like. Photodiode specificinformation may include, for example, diode type, operating parameters,dynamic range, detection limits, filters, wavelength, and the like.Information related to the control system may include, for example,number and placement of motors, number and placement of positionsensors, degrees of freedom, ranges of motion, viable paths, thepresence or absence of a robotic loading system (e.g., robotic system toload each plate or to load each stack tube, etc.), and the like.Information related to the ECL electronics/subsystem may include, forexample, power source, range of waveforms that can be applied, numberand position of electrical contacts, range of motion of electricalcontacts, and the like. Once the configuration file is read 3641, thecomputer system may execute appropriate instructions to set theappropriate motion parameters 3642, set the appropriate detectionparameters 3643, initialize the motion controller and related subsystems3644 (e.g., directing the appropriate subsystems to carry out “home”instructions for the cover axis 3645, contact axis 3646 and carriageaxis 3710) and initialize any other systems or subsystems which may bedefined in the configuration file read at 3641. FIG. 36f is a moredetailed block diagram illustrating the operations carried out at step3710. In one embodiment, homing the carriage axis 3710 might involvemoving the carriage to the home position while remaining at high current3711, moving the contacts to the lock position 3712 and holding thecurrent to the carriage axis 3713.

At step 3602 the user/operator can indicate to the computer system thatinstructions should be executed to prepare for reading the plate 3650.FIG. 36d is a more detailed block diagram illustrating the operationscarried out at step 3650. In one embodiment as show in FIG. 36d ,preparation for reading a plate may include the steps of determiningwhether the cover is open 3651, and if not, allowing a user/operator toindicate that the cover should be opened 3652 (e.g., by pressing thedoor sensor as indicated at step 3623 of FIG. 36b ) and executinginstructions directing the appropriate subsystems to open the cover3653. If the cover was determined to be open at 3651, the computersystem could determine whether an “old plate” is present in the platecarrier 3654 and if so, the computer system may be programmed to promptthe user to unload the old plate 3655. Next a new plate could be loaded3656 by the user/operator and a determination can be made as to whetherthe plate should be read immediately 3657 and if so the user/operatorcould indicate to the system that the plate should be read immediately3662. In the embodiment depicted in FIG. 36d , as a safety precaution,the system could be programmed to determine whether the cover has beenleft open for more than a specified period of time (e.g., thirty (30)minutes) 3660 without any action by the user/operator, and if so executeinstructions directing the appropriate subsystems to close the cover3661.

Once an indication has been received that the plate should be read,e.g., the user could press a button on the device itself or could pressa button displayed by the graphical user interface software,instructions would be executed directing the appropriate subsystems toread the plate 3660. FIG. 36e is a more detailed block diagramillustrating the operations carried out at step 3660. In the embodimentdepicted in FIG. 36e , the computer system could execute instructionsdirecting the appropriate subsystems to home the cover axis (i.e., closethe cover) or ensure that it has been homed (i.e., closed) 3671, applyhigh current to the carriage axis 3672, home the contact axis (i.e.,retract the electrical contacts or ensure that they have been retracted)3673 and move the plate carrier, or carriage, to the first row to beread 3674. Prior to proceeding with the reading process, the plateorientation is determined 3675. At step 3680 instructions are executeddirecting the appropriate subsystems to read the plate by moving theelectrical contacts to the proper plate height 3681 and reading the ECLsignal 3682 from the row of wells under investigation. Next a platetemperature measurement is taken 3683 for use in temperature correctionof the ECL signal and the acquired data is output to, for example, oneor both an electronic file stored on a fixed disk storage device coupledto the computer system or a portable computer readable medium (e.g.,floppy diskette, CD-ROM, DVD, magneto-optical storage medium, or thelike). After the data has been acquired, instructions are executeddirecting the appropriate subsystems to home the contact axis (i.e.,retract the electrical contacts) and a determination is made as towhether there are any remaining unread rows 3676. If the last row hasnot been read, the appropriate subsystems are directed to move thecarriage to the next row 3677 for reading 3680. Once the last row hasbeen read, the appropriate subsystems are directed to home the carriageaxis (i.e., return the carriage to the plate loading/unloading position)3678, as described in greater detail above with reference to FIG. 36c ,step 3710, and open the cover 3679.

In one embodiment, this plate reading procedure 3650, 3660 can becontinuously repeated 3605 for as many iterations as the user/operatorspecifies. If there are no more plates to be read, the user/operator mayindicate that the system should be shutdown 3606. At this point, theuser/operator can unload the plate 3690 and allow the system to beshutdown 3700. Again, the system could be programmed to sit idle for acertain predefined amount of time 3607 (e.g., thirty (30) minutes)before closing the cover, or ensuring that it is closed 3608.

It should be understood that the user/operator steps discussed above forone possible embodiment may be automated as well by, for example,utilizing robotic manipulation systems, and that the invention is notlimited to requiring human intervention for loading/unloading plates,opening the cover, etc.

Image Processing

FIG. 35a depicts an illustrative process flow diagram for one embodimentwhere a CCD camera is used to acquire images of luminescence-basedassays performed in one or more wells of a microplate. FIG. 35e depictsin greater detail the step of reading a plate as depicted in FIG. 35 a.Generally, use of a CCD camera for image acquisition and/or analysistypically requires that certain factors be taken into account and thatcertain measures be taken to insure precision, accuracy and or integrityof the data. Typical factors include CCD chip defect correction,background image subtraction/correction, cosmic ray removal/correction,hardware binning, software binning, image transposition and variousother factors known to those of ordinary skill in the art. Some of thesefactors are often times modified by the unique and/or particularapplication; e.g., background image subtraction/correction whenimaging/analyzing celestial bodies from the earth may depend on certainvariables that are different from imaging/analyzing celestial bodiesfrom outer space. Consequently, and as discussed in greater detailbelow, the general factors affecting use of a CCD camera forimaging/analyzing image data of luminescence-based assays performed inone or more wells of a microplate must be considered in light of thevariables associated with the particular application. Other factorswhich are not present in typical applications, whether modified or not,but instead are present only in the particular application of a CCDcamera for image acquisition/analysis of luminescence-based assaysperformed in one or more wells of a microplate include, for example,creating and applying a well mask, image alignment/centering, averaging,cross-talk correction, dark image detection, saturation detection andother factors which may affect image acquisition/analysis.

In one embodiment, CCD camera defect correction can be based on auser-defined defect map. A defect map could simply be a text-based filewhich defines areas of the camera that are defective or nonfunctioningand that therefore would be excluded from image acquisition/analysis.For example, each defect could be defined as a specific pixel or arectangular area on the full chip image with top-left and bottom-rightcoordinates. A defect map listing each CCD chip defect could, forexample, be determined by analyzing a full chip image under both darkand illuminated conditions. In one embodiment, a defect map may consistof an initial defect map that was created at the time of manufacture ofthe CCD camera and a real-time defect map which is created and updatedsubsequent to CCD camera manufacture. This real-time defect map could,for example, be created upon initial camera installation and thereafterupdated either at regularly scheduled intervals, including weekly,monthly, after a certain number of uses, each use of the camera, or atintervals that are arbitrarily specified or selected by theoperator/user.

The defect maps could be stored in electronic format or innon-electronic format. Electronic format files could either be stored inthe camera hardware, such as for example in the camera firmware, bios,memory registers, or the like, or in an electronic file stored on amachine-readable storage medium that is separate and distinct from thecamera hardware. The separate and distinct electronic file could be adatabase file that is stored as part of the overall system or as andindependent file that is used, for example, as a configuration file orinitialization file. Alternatively, the defect map could be stored in anon-electronic file on traditional non-electronic media and manuallyentered into the system as part of the setup process or procedure. Inembodiments where the defect maps are not stored in the camera hardware,the defect maps could include an identification entry/field that couldenable assignment of the particular camera with a particular set ofdefect maps associated with that camera. Typically, since defect mapsmay be specified in full chip image coordinates, prior to anypre-processing, the defect correction is preferably the first operationperformed on an image. In an illustrative embodiment, a computer couldbe programmed to conduct such image processing automatically.

Where defect correction must be utilized, as for instance when there isan associated defect correction map for a particular camera, correctioncould be achieved by, for example, substituting for each defective pixelvalue an average value of its neighboring pixels. Neighboring values maybe selected from nearest neighbor pixels or from second nearest neighborpixels if common defects include row or column defects. For example, ifa defect is defined as a single pixel I_(r,c), where r represents thepixel's row value and c represents the pixel's column value then thefollowing second nearest neighbor formula could be applied:

$I_{r,c} = \frac{I_{{r - 1},{c - 1 +}}I_{{r - 1},{c + 1 +}}I_{{r + 1},{c - 1 +}}I_{{r + 1},{c + 1}}}{4}$Where a defect is not limited to a single pixel but instead may includean entire column or a portion of a column, then the following formulacould be applied to obtain the corrected value for each pixel in thecolumn or portion of the column:

$I_{r,c} = \frac{I_{r,{c - 1 +}}I_{r,{c + 1}}}{2}$Additionally, where a defect includes an entire row or portion of a row,then the following formula could be applied for each pixel in the row orportion of the row:

$I_{r,c} = \frac{I_{{r - 1},{c +}}I_{{r + 1},c}}{2}$In the event that a defect includes more than a single adjacent row orcolumn or portion of a single adjacent row or column, then thesubstitution value could be obtained through use of, for example, linearinterpolation between its next closest adjacent neighbors.

An embodiment may also require another typical imageprocessing/correction operation which could include, for example, imagetransposition and/or rotation, where for example the particular camerainstallation would result in an image that would not be naturallyoriented for a user/operator. In this instance the image could betransposed and/or rotated to give the user a natural image orientation.

Another factor that typically is considered when using a CCD camera forimage acquisition/analysis is the possibility that one or more pixels ofthe CCD camera may have been impinged upon by a cosmic ray; i.e., acosmic ray hit. Cosmic rays can cause bright spots to occur on thesensor and therefore affect the resulting image acquisition/analysis.Cosmic ray hits occur randomly both in space and time. Therefore, inorder to ensure proper image acquisition/analysis, it is advantageous toidentify pixel values that may be considerably greater than the valuesobserved in a local area of the CCD camera and make corrections asrequired. In one embodiment, a cosmic ray removal/correction algorithmcould be employed that first identifies cosmic ray hits by findingpixels that are brighter then their neighbors, as may be defined by athreshold value (e.g. by using a gradient operation to identify largedifferences in the values of neighboring pixels), or by identifyingpixels that are determined to be statistically significant outliers; Athreshold value may be defined by, for example, a fixed, preset value, afixed, user-specified value, a variable, preset value based on certainvariables or a variable, user-specified value based on certain variablesthat are either predefined or defined by the user. For example, athreshold value may be defined as a factor to be applied to a particularpixel. Therefore, in one embodiment, it may be specified that if apixel's value is four (4) times greater than each of its surroundingneighbor pixels, then it would be considered a cosmic ray hit.Alternatively, a cosmic ray hit may be identified by simply comparing apixel's value with either its neighboring pixels in the same row or itsneighboring pixels in the same column, as opposed to comparing thepixel's value with both its row-adjacent and column-adjacent pixels'values.

Once a pixel value has been identified as likely being the result of acosmic ray hit, the pixel value could be replaced by an average value ofthe neighboring pixels. Such a cosmic ray removal/correction procedurecould be applied repeatedly until an acceptable image results.Additionally, a background value, or offset, could be pre-subtractedfrom all pixels prior to performing the cosmic ray hit search.

For example, a procedure for identifying cosmic ray hits that compareseither the row-adjacent or column-adjacent values could begin by a userspecifying both a threshold value (T=4) and background, or offset value(0-7). Then, for all the pixels in the image the minimum value could beascertained (M). Next, the pixel value offset could be computed usingthe following formula:C=M−ONext, for each image pixel I_(i), the parameters R_(i−1) and R_(i+1),where “i” refers to either the row or column pixel, could be computedusing the following formulas:

$R_{i - 1} = \frac{I_{i} - C}{I_{i - 1} - C}$and

$R_{i + 1} = \frac{I_{i} - C}{I_{i + 1} - C}$Finally, if the parameters R_(i−1) and R_(i+1) are each greater than thethreshold value T, the pixel value is likely the result of a cosmic rayhit. In addition, the cosmic ray removal/correction procedure could beused multiple times to remove/correct the cosmic ray hits that damagemore than one pixel.

Still another factor that typically is considered when using a CCDcamera for image acquisition/analysis is the use of hardware binning toimprove detection limits of the CCD camera. For example, binning of CCDpixels in hardware can be used to reduce read noise per unit area. Readnoise may include noise that results from the process ofanalog-to-digital (A/D) conversion of the analog signal, or the like. Inone embodiment, larger binning could result in lower total electronicnoise, and preferably larger binning is used until the electronic noiseis driven down to a level where the read noise is less than the noisedue to the dark current; dark current is typically unaffected by thebinning choice. In one embodiment, where a CCD camera is used foracquiring/analyzing images of luminescence-based assays performed in oneor more wells of a microplate, binning advantageously has the addedbenefit of faster readout time and reduced image data. Larger binning,however, also may result in reduced dynamic range; i.e., the detectorcould saturate at lower light levels. Binning may also affect theresolution of the images and therefore the level of binning that may beused in a particular application may be limited by that particularapplication's resolution requirements. In certain embodiments, typicalbinning settings could include, for example, 2×2, 4×4, 8×8, and thelike, or no binning at all.

As discussed above, other factors that are not generally found in otheruses of a CCD camera for acquiring/analyzing images ofluminescence-based assays performed in one or more wells of amicroplate, could be taken into consideration. For example, it may bedesirable to ascertain the integrity of the light-tight enclosure byperforming a light leak detection routine. Light leak detection could beperformed by acquiring a background image and using the image statisticsof the background image to determine whether the integrity of thelight-tight enclosure has been compromised. In one embodiment theaverage intensity and standard deviation of the background image couldbe compared with user-defined light leak detection threshold values. Forexample, if either the average value or the standard deviation of thebackground image intensity is greater than the corresponding thresholdvalue, then a light leak condition may exist. The light leak conditioncould, for example, be flagged for subsequent processing, a warningcould be issued to the user/operator, operation could be terminateduntil a user/operator has taken corrective action or affirmativelyindicated that operation should continue despite the light leakcondition, etc. The light leak detection procedure could be carried outprior to performing defect correction and/or cosmic rayremoval/correction, but preferably is performed only after the image hasbeen processed to reduce the potential effects that defects and/orcosmic ray hits may have on identification of a light leak condition.

Another factor which could be taken into consideration relates toresizing of the image(s) acquired. In some embodiments, in order to makeimage processing independent of any hardware binning, and in some casesto reduce processing time, both the background and sector images couldbe resized to certain predefined sizes. For example, a predefined sizecould be 160×160 for a 96 well plate, 320×320 for a 384 well plate,640×640 for a 1536, and 640×640 for multi-spot plates. Where resizing isused and where the original image size is greater than the predefinedsize, the resized image pixel value could be a sum of the sub-pixels,for example:

${{factor} = \frac{original}{predefined}},{and}$$I_{ne{w{({r,c})}}} = {\sum\limits_{i,{j = 0}}^{factor}I_{{{original}{({{{{rx}\mspace{11mu}{factor}} + 1},{{{cx}\mspace{11mu}{factor}}\; + \; j}})}},{{{{where}\mspace{14mu}{original}\mspace{14mu}{size}} > {{predefined}\mspace{14mu}{size}}};}}}$

Alternatively, if the original image size is smaller than the predefinedsize, the resized image pixel value could be obtained by distributingthe original image pixel value among N pixels of the resized image. Inother words, to calculate the resized image pixel value, the originalimage could be unfolded iteratively by a factor of two (2) until theresized image equals the predefined size. For example, upon eachiteration, the intermediate pixel value could be calculated based uponone quarter (¼) of the original pixel value and a weighted value for itsneighboring pixel values. The original pixel could, for example be givena weight of nine sixteenths ( 9/16), the next row-adjacent pixel couldbe given a weight of three sixteenths ( 3/16), the next column-adjacentpixel could be given a weight of one sixteenth ( 1/16), and thediagonal-adjacent pixel could be given a weight of one sixteenth (1/16), for example:

${factor} = {\frac{predeftned}{original}\mspace{14mu}{and}}$${N = {{factor}^{2}\mspace{11mu}{then}}},{I_{{intermediate}{({r,c})}} = {{9\text{/}16 \times 1\text{/}4\mspace{14mu}{X_{original}^{I}\left( {\frac{r}{2},\frac{c}{2}} \right)}} + {3\text{/}16 \times 1\text{/}4\mspace{14mu}{X_{origianl}^{I}\left( {{\frac{r}{2} - 1},\frac{c}{2}} \right)}} + {3\text{/}16 \times 1\text{/}4\mspace{14mu}{X_{original}^{I}\left( {\frac{r}{2},{\frac{c}{2} - 1}} \right)}} + {1\text{/}16 \times 1\text{/}4\mspace{14mu}{X_{original}^{I}\left( {{\frac{r}{2} - 1},{\frac{c}{2} - 1}} \right)}}}}$$I_{{intermediate}{({{r + l},c})}} = {{9\text{/}16 \times 1\text{/}4\mspace{14mu}{X_{original}^{I}\left( {\frac{r}{2},\frac{c}{2}} \right)}} + {3\text{/}16 \times 1\text{/}4\mspace{14mu}{X_{origianl}^{I}\left( {{\frac{r}{2} + 1},\frac{c}{2}} \right)}} + {3\text{/}16 \times 1\text{/}4\mspace{14mu}{X_{original}^{I}\left( {\frac{r}{2},{\frac{c}{2} - 1}} \right)}} + {1\text{/}16 \times 1\text{/}4\mspace{14mu}{X_{original}^{I}\left( {{\frac{r}{2} + 1},{\frac{c}{2} - 1}} \right)}}}$$I_{{intermediate}{({r,{c + l}})}} = {{9\text{/}16 \times 1\text{/}4\mspace{14mu}{X_{original}^{I}\left( {\frac{r}{2},\frac{c}{2}} \right)}} + {3\text{/}16 \times 1\text{/}4\mspace{14mu}{X_{origianl}^{I}\left( {{\frac{r}{2} - 1},\frac{c}{2}} \right)}} + {3\text{/}16 \times 1\text{/}4\mspace{14mu}{X_{original}^{I}\left( {\frac{r}{2},{\frac{c}{2} + 1}} \right)}} + {1\text{/}16 \times 1\text{/}4\mspace{14mu}{X_{original}^{I}\left( {{\frac{r}{2} - 1},{\frac{c}{2} + 1}} \right)}}}$$I_{{intermediate}{({{r + l},c})}} = {{9\text{/}16 \times 1\text{/}4\mspace{14mu}{X_{original}^{I}\left( {\frac{r}{2},\frac{c}{2}} \right)}} + {3\text{/}16 \times 1\text{/}4\mspace{14mu}{X_{origianl}^{I}\left( {{\frac{r}{2} + 1},\frac{c}{2}} \right)}} + {3\text{/}16 \times 1\text{/}4\mspace{14mu}{X_{original}^{I}\left( {\frac{r}{2},{\frac{c}{2} + 1}} \right)}} + {1\text{/}16 \times 1\text{/}4\mspace{14mu}{X_{original}^{I}\left( {{\frac{r}{2} + 1},{\frac{c}{2} + 1}} \right)}}}$

Yet another factor which could be taken into consideration relates tobackground subtraction. CCD cameras integrate thermally generatedelectrons as well as electrons that result from exposure to a lightsource. In one embodiment, a background image taken with the samesettings as the luminescence based assay image can be subtracted inorder to cancel the potentially adverse effects that thermally generatedelectrons may have on the acquisition/analysis of images fromluminescence based assays. The subtraction of the background image canbe accomplished by using a differencing operation to remove thecontribution of the thermally generated electrons. This could simply bethe subtraction of the background image array from the actual imagearray, for example:I _(new(r,c)) =I _(original(r,c)) −I _(background(r,c))

Still another factor which could be taken into consideration relates tocreating and applying an image mask that corresponds to the particularmicroplate layout being imaged. In one embodiment, the image mask couldbe a binary matrix M_((r,c)) of the same size as the acquired image. Thebinary matrix could define pixels that are in optical registration withthe wells of a microplate or with one or more spots within a well, witha value of one (1). In addition, where manufacturing defects introducemisalignments, the image mask could also be rotated relative to acertain coordinate on the microplate by adding offsets to the centers'coordinates. Once the image mask has been created, the image mask can byapplied simply by multiplying the acquired image matrix and the binaryimage mask matrix.

In one embodiment, the user could be required to specify certainparameters in order to create the appropriate image mask for aparticular microplate configuration. For example, a user could specifythe following parameters to define the plate configuration: the platetype that defines the number of wells in the mask; well radius and wellspacing that could be in absolute units or in units as a function of thepredefined image size; well shape, such as circle, square, or the like;and the coordinates of the center mask in, for example, column (X) androw (Y) coordinates. For multi-spot plates the user could also berequired to define the spacing between spots in the well, thearrangement of spots in the well, the size of the spots in the well, andthe like. In another embodiment, one or more of the previously describedparameters could be automatically specified by an indicator found on theplate itself, for example, through use of a bar-code label placed on theplate at the time of manufacture or at the time the reagents areapplied.

In certain instances, another form of misalignment or error may bepresent as evidenced when the actual sector image does not perfectlyalign with the center of the entire image taken by the CCD camera. Suchmisalignment may be the result of, for example, mechanical changes inthe instrument itself, mis-registration of the microplate beneath theCCD camera, and the like. In one embodiment, such misalignment may betaken into consideration by performing one or more of calibrating theinstrument to set the coarse center position and angle after anymechanical change to the instrument and performing a fine adjustment oneach sector. Calibration of the instrument may be performed by theuser/operator or by service technicians and may be performed accordingto a predefined maintenance schedule, upon the occurrence of a certainmaintenance event, after a predefined number of uses, or the like. Thiscalibrated center could then be used to position the microplate beneaththe CCD camera through the use of for example, stepper motors.

In one embodiment, fine adjustment can be accomplished through acomputer-implemented process that automatically performs a fineadjustment of the image mask alignment on every sector to compensate forany slight plate mis-registration. For example, an auto-centeringalgorithm could be used that calculates a correlation function F(Δr, Δc,Δθ), where Δr, Δc, and Δθ are positional and rotational offsets betweenthe mask and image, to locate the actual center and rotation of themicroplate sector. Proper calibration, as discussed above, would help toensure that, starting at the calibrated position, the closest localmaximum in the correlation function will be the true center. To find themaximum, the correlation function could be calculated for all possibleΔr, Δc, and Δθ, or alternatively, an iterative process can be used tolocate the maximum by taking steps in Δr, Δc, and Δθ towards increasingvalues of F(Δr, Δc, Δθ).

In one embodiment the auto-centering algorithm could begin by usingreduced-resolution images to get the initial aligned position, and thenprogress through several steps of higher resolution images to home in onthe precisely aligned position. Such an approach could provide for rapidconvergence to the optimally aligned position. For example, in a firststep the auto-centering algorithm could begin with a low resolutionimage by binning the image four (4) fold, evaluate the correlationfunction F(Δr, Δc, Δθ) at values of Δr, Δc, Δθ that are offset by onepixel and angle increment, and then move to the position at which F isgreatest. The process would then repeat until the maximum value of thecorrelation function is found, or until some predefined number ofiterations has been carried out. In one embodiment, it could bespecified that if the maximum is not found within the predefined numberof iterations an error message could be displayed and/or the image couldbe checked for being dark. If the maximum is found then thecorresponding center becomes the starting point for the next step, atwhich point the image would be unfolded by a factor of two (2) and theprocedure is repeated until the initial (predefined) image size isreached.

In order to insure that a few very bright wells do not dominate thealignment calculation, in one embodiment, a normalizing function can beapplied to the image to more evenly weight the bright and dimspots/wells. One example of such a normalizing function consists oftaking the third root of all pixel values in the image.

Finally, the center that corresponds to the greatest value of F(Δr, Δc,Δθ) would be considered to be the true center. Additionally, the newlycalculated center could be compared to the initial calibrated center andif it is determined that the new center is more than a predefined value,such as % of the well/spot spacing, a precautionary measure could betaken such as issuing a warning, halting operation of the instrument, orthe like.

As discussed above, if the auto-centering algorithm cannot find the truecenter of the sector image within a certain predefined number ofiterations, then another procedure can be used to determine if the imageis actually dark. Alternatively, identification of a dark image could beaccomplished prior to carrying out the steps outlined above forauto-centering. In one embodiment, dark image detection can beaccomplished by comparing the result values with certain predefinedbaseline values. The baseline values can be empirically defined withreference to, for example, typical values of assay buffer results, platetype, and the like. For example, if the maximum result value of a sectoris less than the baseline value then the image would be considered to bedark.

Still yet another factor that could be taken into consideration relatesto cross-talk. Cross-talk occurs due to optical system imperfectionssuch as, for example, when light from one well, or spot, diffracts,refracts, is multiply reflected or scattered (“bleeds”) into aneighboring well, or onto a neighboring spot. In one embodiment,cross-talk can be empirically measured and a corrective matrix can beassembled and used to deconvolve the cross-talk. The cross-talkcorrective matrix is the inverse of the cross-talk matrix between awell, or a spot, and its neighbors. Cross-talk correction can be appliedto single spot and multispot applications, however, experience has shownthat satisfactory performance can be achieved in single spotapplications without cross-talk correction.

In addition to cross-talk correction, in certain embodiments it may beadvantageous to provide for correction of collection efficiencyvariations that may be present across a well. In certain embodiments,the collection efficiency across a well may not be uniform. For example,light from regions near the edge of the well may not be as efficientlycollected as from the region near the center of the well. In embodimentswhere multiple spots are located in a single well, for example,variation in collection efficiency could be more significant since it ispossible that the same reaction located at a region near the edge of awell could appear to have a lower pixel value. In embodiments where onlysingle spots are used in each well variations in collection efficiencymay still be present since it could be possible that the same reactionlocated at a region near the edge of a plate could appear to have alower pixel value. In one embodiment, such variations in collectionefficiency could be corrected by, for example, calculating orempirically determining the efficiencies and using an appropriate scalefactor to the initial reported pixel values measured.

Still further corrections may be necessary to account for thermalsensitivity/variation. It has generally been observed thatluminescence-based assays may exhibit some thermaldependency/sensitivity. In one embodiment, it would be advantageous tocontrol and/or measure the temperature and/or any variations in order toachieve optimal results. Temperature variations may exist, for example,across a microplate, in one or more localized portions of a microplate,within a single well, or the like. Measuring temperature and/ortemperature variations may be accomplished by, for example, utilizingeither a contact or non-contact temperature sensor. Contact temperaturesensors may include thermistors, thermocouples, and the like and wouldbe used to measure temperature of for example, the bottom of themicroplate in order to estimate the actual temperature of the reactantswhereas non-contact temperature sensors may include infraredphotometers, infrared spectrometers, lasers, and the like and would beused to take remote temperature readings of the reactants.

Thermal corrections may be applied by, for example, using empiricalrelations derived from data for a particular assay's and/or label'stemperature dependence to determine the appropriate thermal correctionfactor which should be applied to a particular set of assay results. Thethermal correction factor may be a single factor that is based-on anaverage temperature for an entire microplate or may be a variable factorwhich could depend on, for example, the actual temperature within eachwell, the actual temperature of a single spot's reactants, the averagetemperature of a single sector, and the like.

In another embodiment, non-contact sensors for remotely detecting boththe temperature of the reactants within one or more wells as well asremotely detecting the temperature at one or more locations on themicroplate itself might be employed to better estimate the actualtemperature of the reactants within a well. The plate could be movedinto a position that would allow the temperature sensor to take ameasurement, the plate could be held in a fixed position and the sensormoved into the measurement position, and/or the plate could be held in afixed position and a scanning sensor could be used to take thermalmeasurements. A scanning sensor could include, for example, the use of anon-contact sensor such as a infrared spectrometer that uses adjustablemirrors to scan various location on the plate and within the wells.

In addition to the previously described image processing procedures, theuse of a CCD camera may also allow for the analysis of the image todetermine, for example, non-uniformity of the reaction, quality of theassay or image acquisition, and the like. In one embodiment, it would bepossible to determine the percentage of pixels that are active to detectnon-uniformity of the reaction which may indicate that there areproblems with the plate and/or the reactants' preparation. In addition,image statistics within one or more wells (e.g., mean, variance, median)could provide some indication of the quality of the read.

According to another preferred embodiment, the apparatus is adapted toallow for image acquisition of the plate prior to inducing ECL todetermine (a) the position of the plate (e.g., centering, location ofspots, etc.), (b) the orientation of the plate (e.g., 180° orientation),(c) type of plate, (d) existence of plate cover or seal, (e) focus, (f)well sample volume, etc. Preferably, the apparatus further comprises aLED or other light source to allow image acquisition of the plate withinthe light tight enclosure, where the light source may be pulsed on toilluminate the plate only during this image acquisition and turned offfor the subsequent measurement of the luminescence. According to afurther embodiment, information gathered from the images acquired withthe light on, prior to inducing ECL, is used as input for subsequentdata processing (e.g., knowledge of the centering of the sector, plateorientation, and plate type).

FIG. 36a depicts an illustrative process flow diagram for one embodimentwhere one or more photodiodes are used to detect luminescence fromluminescence-based assays performed in one or more wells of amicroplate. FIG. 36e depicts in greater detail the step of readingluminescence as depicted in FIG. 36a . Preferably, the flow diagramfurther comprises the step of measuring the plate and/or sampletemperature. Use of one or more photodiodes to detect luminescence fromone or more wells of a microplate could require consideration of certainadditional/modified factors, and implementation of certainadditional/modified corrective measures, beyond those previouslydiscussed with reference to CCD cameras. For example, backgroundprediction and subtraction when using photodiode sensors varies sincethe luminescence data acquired using photodiode sensors is in the formof a waveform. Of course it should be understood that the mode of use ofa CCD cameras, as discussed above, is not limited to integrating the ECLsignal in the detection hardware itself but alternatively may be used ina mode where the CCD camera measures the ECL signal intensity as afunction of time and integrate the signal in a programmable computersystem programmed with the appropriate algorithm(s) and set of softwareinstructions. In one embodiment, luminescence data is acquired bothbefore and after activation of the luminescence-based assays in order toobtain background readings that represent the dark condition. The darkvalues are acquired before and after the activation of theluminescence-based assays to remove the effect of electronic drift,i.e., low frequency noise, and offset. An estimate of the dark signalcan be generated using both measurements and a linear, quadratic orother model could be used to correct for any background light that mayoriginate from, for example, the plate when a white microplate is usedto increase collection efficiency (for example, due to phosphorescence).The dark signal estimate is subtracted from the measured signal and theresulting waveform is integrated in time to obtain the final reading.Alternatively, rather than integrating, a known temporal responsefunction can be used to fit the measured response.

Alternatively, in another embodiment where the luminescence-based assayis, for example, an ECL assay, the activation waveform could be pulsedand the dark signal could be measured between one or more pulses. Suchpulsing could, for example, improve the detection limits by more aptlyremoving low-frequency noise by essentially shifting the signal to ahigher frequency.

5.11 Method of Selecting Biologically Active Compounds and ProducingNovel Drugs

Another aspect of the invention relates to improved methods and systemsfor selecting or identifying biologically active compounds and,optionally, incorporating such biologically active compounds intosuitable carrier compositions in appropriate dosages. The inventionincludes the use of the multi-well plates, apparatuses, systems, kitsand/or methods of the invention to screen for new drugs, preferably, byhigh-throughput screening (HTS), preferably involving screening ofgreater than 50, more preferably 100, more preferably 500, even morepreferably 1,000, and most preferably 5,000. According to a particularlypreferred embodiment, the screening involves greater than 10,000,greater than 50,000, greater than 100,00, greater than 500,000 and/orgreater than 1,000,000 compounds.

One embodiment of the invention relates to a method for selecting oridentifying biologically active compounds from a library of compounds,said method comprising screening said library of compounds forbiological or biochemical activity, wherein said screening includesassaying the library of compounds for the biological or biochemicalactivity, the assays being conducted using the plates and/or apparatusof the invention.

Preferably, the method further comprises identifying one or more activecompounds.

Preferably, the method further comprises testing said one or more activecompounds for bioavailability, toxicity and/or biological activity invivo. According to one preferred embodiment, the testing comprisesfurther screening using the plates and/or the apparatus of theinvention.

Preferably, the method further comprises synthesizing analogues of saidone or more active compounds. According to one preferred embodiment, theanalogues are screened for bioavailability, biological activity and/ortoxicity using the plates and/or apparatus of the invention.

According to a particularly preferred embodiment, the method furthercomprises formulating the one or more compounds into drugs foradministrating to humans and/or animals. Preferably, the formulatingcomprises determining the suitable amount of the one or more activecompounds in the drug and mixing the suitable amount with one orexcipients or carriers. Preferably, the excipient comprises sugar and/orstarch.

Another embodiment of the invention relates to a method of analyzing acomplex mixture of biochemical substances to measure a plurality ofbinding components therein, comprising:

(a) introducing said mixture into a multi-well plate adapted forelectrode induced luminescence assays (preferablyelectrochemiluminescence assays), said plate comprising a plurality ofwells having a plurality of binding reagents therein;

(b) inducing one or more samples in said wells to luminesce; and

(c) measuring the luminescence from each of said wells.

Another embodiment of the invention relates to a method of analyzing theoutput of a combinatorial (biological and/or chemical) mixture tomeasure a plurality of binding components therein, comprising:

(a) introducing said mixture into a multi-well plate adapted forelectrode induced luminescence (preferably electrochemiluminescence)assays, said plate comprising a plurality of wells having a plurality ofbinding reagents therein;

(b) inducing one or more samples in said wells to luminesce; and

(c) measuring the luminescence from each of said wells.

Another embodiment of the invention relates to a method for measuring asingle biochemical substance in a sample in a multiplicity ofsimultaneous assays, comprising:

(a) introducing said sample into a multi-well plate adapted forelectrode induced luminescence (preferably electrochemiluminescence)assays, said plate comprising a plurality of wells having a plurality ofbinding reagents therein;

(b) inducing one or more samples in said wells to luminesce; and

(c) measuring the luminescence from each of said wells.

A further embodiment of the invention relates to a method of drugdiscovery comprising:

(a) selecting a multiplicity of compounds for testing;

(b) screening said multiplicity of compounds for biological activity(using any one of the multi-well plates and/or apparatus describedabove) to find one or more biologically active compounds; and

(c) modifying said one or more biologically active compounds to reducetoxicity and/or enhance biological activity thereby forming one or moremodified biologically active compounds.

Preferably, the method further comprises screening said modifiedbiologically active compounds for biological activity and/or toxicity(using the multi-well plate and/or apparatus described above).

Preferably, the method further comprises determining the appropriatedosage of one or more of said modified biologically active compounds.Preferably, the method still further comprises incorporating such dosageinto a suitable carrier such as sugar or starch to form a drug in solid(e.g., pill or tablet) or liquid form.

Advantageously, the methods, apparatus and/or assay plates or modules ofthe invention may be integrated into and/or used in a variety ofscreening and/or drug discovery methods. Such screening and/or drugdiscovery methods include those set forth in U.S. Pat. No. 5,565,325 toBlake; U.S. Pat. No. 5,593,135 to Chen et al.; U.S. Pat. No. 5,521,135to Thastrup et al.; U.S. Pat. No. 5,684,711 to Agrafiotis et al.; U.S.Pat. No. 5,639,603 to Dower et al.; U.S. Pat. No. 5,569,588 to Ashby etal.; U.S. Pat. Nos. 5,541,061; 5,574,656; and U.S. Pat. No. 5,783,431 toPeterson et al.

According to another embodiment, the invention further comprisesidentifying adverse effects associated with the drug and storinginformation relating to the adverse effects in a database. See, U.S.Pat. No. 6,219,674 by Classen, hereby incorporated by reference.

Another aspect of the invention relates to improved biologically activecompounds and/or drugs made using the inventive methods.

The following examples are illustrative of some of the apparatuses,plates, kits and methods falling within the scope of the presentinvention. They are, of course, not to be considered in any waylimitative of the invention. Numerous changes and modification can bemade with respect to the invention by one of ordinary skill in the artwithout undue experimentation.

6. EXAMPLES 6.1 Fabrication of Multi-Well Assay Plates Having ScreenPrinted Electrodes

Multi-layer plate bottoms were prepared by screen printing electrodesand electrical contacts on 0.007″ thick Mylar polyester sheet. The Mylarsheet was first cut with a CO₂ laser so to form conductive through-holes(i.e., holes that were subsequently made conductive by filling withconductive ink) as well as to form alignment holes that were used toalign the plate bottom with the plate top. Electrical contacts wereformed on the bottom of the Mylar sheet by screen printing anappropriately patterned silver ink layer (Acheson 479ss) and a carbonink overlayer (Acheson 407c). The carbon ink layer was dimensionedslightly larger (0.01 inches) than the silver ink layer to preventexposure of the edge of the silver film. Working and counter electrodeswere formed on the top of the Mylar film in a similar fashion exceptthat three layers of carbon ink were used to ensure that no silverremained exposed. The conductive through-holes filled with conductiveink during these screen-printing steps. A dielectric ink wassubsequently printed over the electrode layers so as to define theactive exposed surface area of the working electrode. Typically, nineplate bottoms were simultaneously printed on an 18″x12″ Mylar sheet.Typical registrational tolerances during the screen printing steps were+/−0.007-0.008 inches on the top side of the substrate and +/−0.010inches on the bottom side. The separation between the printed counterand working electrode strips was kept at >0.010 inches to prevent theformation of short circuits. The working electrodes were conditioned foruse in assays by treating the patterned plate bottoms for 5 min. with anoxygen plasma (2000 W, 200 mtorr) in a plasma chamber (Series B,Advanced Plasma Systems, St. Petersburg, Fla.) modified with large areaflat electrodes.

Multi-well assay plates were assembled using the plate bottoms describedabove and injection molded plate tops. The dimensions of the plate topsmet industry standards as established by the Society of BiomolecularScreening. The plate tops were either made of black plastic (polystyreneloaded with black pigment) or white plastic (polystyrene loaded withtitanium dioxide). The bottom surfaces of the plate tops were contactedwith die-cut double sided tape (1 mil PET coated on each side with 2 milof acrylic pressure sensitive adhesive) so as to allow for sealing ofthe plate tops to the plate bottoms. The tape was cut to form holes thatwere slightly oversized relative to the holes in the plate tops. Theplate bottoms were fixed (using the laser cut alignment holes) ontoalignment pins on an X-Y table. The plate bottoms were optically alignedto the plate tops and then sealed together using a pneumatic press (400pounds, 10 s). Alignment was carried out sufficiently accurately so thatthe exposed working electrodes were centered within the wells (+/−0.020inches for 96-well plates and +/−0.015 inches for 384 well plates).These tolerances ensured that the exposed regions of the workingelectrodes were within the wells and that there were exposed counterelectrode surfaces on both sides of the working electrode.

A variety of types of multi-well assay plates were prepared according tothe procedure described above. A few specific plate designs aredescribed in more detail below to allow for reference in subsequentexamples. Plate A, a 96-well plate sectioned into 12 columnar sectors of8 wells, was prepared using components and patterns as pictured in FIGS.10A-10B and a white plate top. Plate B, a 96-well plate sectioned into 6square sectors of 4×4 wells, was prepared using components and patternsas pictured in FIG. 11A and a black plate top. Plate C, a 96-well platesectioned into 6 square sectors of 4×4 wells, was prepared usingcomponents and patterns as pictured in FIGS. 14A-14B (except that theelectrodes and contacts are sectioned such as illustrated in FIG. 11A(Details A and C)) and a black plate top. The dielectric layer in PlateC is patterned so as to expose four isolated “fluid containment regions”on the working electrode surface within each well. Plate D was similarto Plate C except that the dielectric layer was patterned so as toexpose 7 isolated “fluid containment regions” on the working electrodewithin each well. Plate E was similar to Plate C except that thedielectric layer was patterned so as to expose 10 isolated “fluidcontainment regions” on the working electrode within each well. Plate F,a 384-well plate sectioned into 6 square sectors of 8×8 wells, wasprepared using components and patterns as pictured in FIG. 12A and ablack plate top. In each of the FIGS. 10A-10B, 11A, 12A and 14A-14B,details A, B, C and D show, respectively, the printed contact layer, theMylar film with through-holes, the printed electrode layer and theprinted dielectric layer.

6.2 Fabrication of Multi-Well Assay Plates Having Plate Bottoms Formedfrom Extruded Carbon-Polymer Composites

This example describes the fabrication of an embodiment of multi-wellassay plate 800 shown in FIG. 8A (referred to hereafter as Plate G). Inthis example, conductive layer 820 was a composite comprising carbonfibrils dispersed in ethylene-vinyl acetate (EVA) copolymer; conductivetape 810 was a conductive foil laminate (Lamart APS-25 having a 0.36 milaluminum film on a 1 mil polyester (PET) substrate coated with a 1 millayer of acrylic pressure sensitive adhesive and having a protectingbacking to protect the adhesive during processing steps); adhesive layer806 was a double-sided adhesive tape comprising a 1 mil polyester film(PET) coated on both sides with 2 mil of acrylic pressure sensitiveadhesive and plate top 802 was an injection-molded black polystyreneplate top that conformed to the Society of Biomolecular Screeningguidelines.

The carbon fibril-EVA composite was prepared as described in PublishedPCT Application WO98/12539 and extruded into a 0.010″ thick sheet. Thesheet was backed with an adhesive polyester liner and cut with a flatbed engraved die (the depth of the cuts were designed so as to leave thesix square sections of the composite in correct orientation on a singlepiece of liner). The conductive tape was die cut using a rotary die andmarried to the top surface of the composite sheet. The exposed topsurface of the composite sheet was conditioned for use in assays bytreating the patterned plate bottoms for 5 min. with an oxygen plasma(2000 W, 200 mTorr) in a plasma chamber (Series B, Advanced PlasmaSystems, St. Petersburg, Fla.) modified with large area flat electrodes.The plate bottom was attached to the plate top using a double sidedadhesive before the bottom was painted with silver and the counterelectrode was folded over. The liner was then removed from the back ofthe composite sheet, the back of the composite sheet was painted withsilver paste and the conductive tape folded around and married to theback of the composite sheet to form the completed plate bottom. Twoother plates were prepared using analogous protocols except: Plate H hada 16×24 arrangement of wells and Plate I was prepared using a 8×12arrangement of wells, but had a white plate top and a fibril compositethat was sectioned into 12 columnar sections as shown in FIG. 8B.

6.3 ECL Measurements

ECL was induced from multi-well assay plates and measured using one oftwo instrumental configurations. Plates that were sectioned into 12columnar sectors of 8 wells (Plates A and H) were read on an instrumentdesigned to make electrical contact to single columnar sectors. Thesector in electrical contact with the instrument was aligned with anarray of 8 photodiodes that were used to measure the ECL emitted fromeach well. A translation table was used to translate the plate under thearray of photodiodes so as to allow all 12 sectors to be read. Platesthat were sectioned into 6 square sectors (Plates B, C, D, E, F and G)were read on an instrument designed to make electrical contact toindividual square sectors. The sector in electrical contact with theinstrument was aligned with a telecentric lens (having a front elementwith a diameter of 4.1″) coupled to a cooled CCD camera (VersArray:1300F, Princeton Instruments) that was used to image ECL emitted fromthe sector. The camera employed a CCD chip with dimensions of roughly2.6 cm×2.6 cm and having a 1340×1300 array of pixels. The pixel size was0.02 mm×0.022 mm. An optical band pass filter in the optical path wasused to select for light matching the emission profile ofruthenium-tris-bipyridine. A translation table was used to translate theplate under the telecentric lens so as to allow all 6 sectors to beread. Image analysis software was used to identify wells or assaydomains within wells and to quantitate ECL from specific wells ordomains. ECL from plates having screen printed carbon working electrodeswas induced using a linear voltage scan from 2.5 V to 5.5 V over 3seconds. ECL from plates having fibril-EVA composite electrodes wasinduced using a linear voltage scan from 2 V to 5 V over 3 seconds. ECLis reported as the total integrated light signal measured over theperiod of the voltage scan (after correcting for background light levelsand detector offset). ECL signals measured on the two differentinstruments are not directly comparable.

6.4 ECL from Ruthenium-Tris-Bipyridine in Solution

Solutions containing varying concentrations ofruthenium(II)-tris-bipyridine dichloride were prepared in a buffercontaining approximately 100 mM tripropylamine and 0.1% triton X-100 in200 mM phosphate buffer, pH 7.5 (Origen® Assay Buffer, IGENInternational). ECL from these solutions was measured (according to theprocedures described in Example 6.3) in multi-well assay plates preparedaccording to Examples 6.1 and 6.2. The volume of solution in the wellswas 100 uL for 96-well plates and 40 uL for 384 well plates. FIGS. 24and 25 show the ECL signal as a function of the concentration ofruthenium-tris-bipyridine in a variety of different plateconfigurations. The plots show that the multi-well assay plates weresuitable for the highly sensitive detection of ruthenium-tris-bipyridinein solution.

6.5 ECL Immunoassay Using Multi-Well Assay Plates

The following example illustrates the use of multi-well assays plates inECL-based sandwich immunoassays. Plates prepared according to Examples6.1 and 6.2 were coated with a capture antibody specific for an epitopeon the analyte of interest. The coating was achieved by dispensing asolution containing the antibody onto the active working electrodesurface of each well and allowing the solution to dry over the course ofan hour. The volume of the solution was chosen so that the antibodysolution would spread over the surface of the working electrode butwould be confined to the surface of the working electrode (i.e., by thephysical barrier provided by either the conductive tape layer or aprinted dielectric layer). The concentration of antibody was chosen soas to provide a small excess of antibody relative to the amount presenton a fully saturated working electrode surface. In some cases it wasfound that the deposition of antibody was more reproducible and/orefficient when a biotin-labeled antibody was used and when the biotinantibody was mixed with avidin prior to deposition on the workingelectrode surface. Alternatively, avidin could be adsorbed on theworking electrode and the biotin-labeled antibody could be bound to theavidin layer in a subsequent step. After drying the antibody solution onthe working electrode, the excess unbound antibody was removed (anduncoated surfaces blocked) by filling the wells with a solutioncontaining 5% (w/v) bovine serum albumin (BSA) in phosphate bufferedsaline (PBS). The plates were incubated with the blocking solutionovernight at 4° C. and then washed with PBS. In this step and insubsequent steps the volume of fluid in the well was sufficient to coverthe entire bottom surface of the well, as opposed to being confined tothe exposed surface of the working electrode. The assays were carriedout by combining in the wells of the coated plates i) the samples andii) a solution (a buffered electrolyte containing BSA, detergent and/orother blocking agents) containing a detection antibody (labeled byreacting with a sulfonated derivative of ruthenium-tri-bipyridine, NHSester 1 shown below) that was specific for a second epitope on theanalyte of interest. The label is described in copending U.S. patentapplication Ser. No. 09/896,974, entitled “ECL Labels Having ImprovedNon-Specific Binding Properties, Methods of Using and Kits Containingthe Same”, filed Jun. 29, 2001, the disclosure hereby incorporated byreference.

The plates were incubated for 1 h at room temperature (96 well plateswere mixed using a plate shaker; the 384 plates were not mixed). Thewells were washed with PBS. The wells were filled (100 uL in 96-wellplates; 40 uL in 384-well plates) with tripropylamine-containingsolution (ORIGEN Assay Buffer, IGEN International) and analyzed usingECL-detection as described in Example 6.3.

FIGS. 26 and 27 show ECL signal as a function of the concentration ofprostrate specific antigen (PSA) in samples as measured on a variety ofdifferent plate configurations. The capture and detection antibodieswere prepared by labeling the same antibodies as used in the RocheElecsys PSA Assay Kit (Roche Diagnostics). FIG. 28 shows ECL signal as afunction of the concentration of alpha-fetoprotein (AFP) as measuredusing an ECL immunoassay for AFP. The capture and detection antibodieswere prepared by labeling the same antibodies as used in the RocheElecsys AFP Assay Kit (Roche Diagnostics). The reported ECL signals arecorrected for background signals as measured using samples that do notcontain the analyte of interest.

6.6 Multi-Analyte Immunoassays in Multi-Well Assay Plates

Sandwich immunoassays for four different cytokines—interleukin 1β(IL-1β), interleukin 6 (IL-6), interferon γ (IFN-γ) and tumor necrosisfactor α (TNF-α)—were carried out simultaneously in the wells of platesmanufactured according to the design and procedure described for Plate Cin Example 6.1, except that antibodies were adsorbed onto the surfacesof the working electrodes subsequent to the plasma treatment step andprior to attachment of the plate top. This plate design has a dielectricpattern printed over the working electrode in each well that exposesfour “fluid containment regions” over each electrode. Four captureantibodies (each selective for one of the analytes of interest) werepatterned into distinct assay domains by microdispensing solutions ofthe antibodies on the fluid containment regions within each well (oneantibody per region) and allowing the antibodies to adsorb to thesurface of the working electrode. Solutions (0.25 uL) containing theantibody (at a concentration of 32 ug/mL for IL-1 and TNF-α or 64 ug/mLfor IL-6 and IFN-γ) and 0.1% BSA in phosphate buffered saline weredispensed onto the fluid containment regions using a solenoid valvecontrolled microdispensor (Biodot Dispensor, Cartesian Technologies) andallowed to evaporate to dryness. The volume of the antibodies wassufficient to spread over all of the exposed electrode surface within afluid containment region but was small enough so that the fluid did notspread past the boundary formed by the dielectric layer. After dryingthe antibody solution on the working electrode, the plate tops wereattached and the excess unbound antibody was removed (and uncoatedsurfaces blocked) by filling the wells with a solution containing 5%(w/v) bovine serum albumin (BSA) in phosphate buffered saline (PBS). Theplates were incubated with the blocking solution overnight at 4° C. andthen washed with PBS.

The assays were carried out by the steps of i) adding 0.02 mL of thesample to the well and incubating for 1 hour on a plate shaker, ii)washing the wells with PBS; iii) adding 0.02 mL of a solution containing2,000 ng/mL each of four detection antibodies (labeled with NHS ester 1)against the four analytes of interest and incubating for 1 hour on aplate shaker, iv) washing with PBS; v) introducing 0.1 mL of a solutioncontaining tripropylamine in phosphate buffer (ORIGEN Assay Buffer, IGENInternational) and vi) measuring ECL as described in Example 6.3. TheECL emitted from the plates was imaged using a cooled CCD camera. Theapparatus used image analysis software to identify the assay domains inthe ECL image and to quantify the light emitted from each of the fourassay domains in each well. FIG. 29 demonstrates that each of theanalytes of interest can be independently measured in a single sample ina single well of a multi-well assay plate. The figures show ECL emittedfrom each assay domain as a function of the concentration of eachanalyte. The introduction of a specific analyte led to a linear increasein ECL with analyte concentration (relative to the background signalmeasured in the absence of any analyte) at assay domains having captureantibodies directed against that analyte, but did not affect the ECL atassay domains having antibodies directed against the other analytes.FIG. 30 shows an image of the ECL emitted from a sector of wells used toassay solutions containing mixtures of the four analytes. Thehighlighted well is annotated to show the arrangement of the four assaydomains. That specific well was used to assay a sample having 250 pg/mLeach of IL-1β and TNF-α and 8 pg/mL each of IL-6 and IFN-γ.

6.7 ECL-Based Nucleic Acid Hybridization Assays in Multi-Well AssayPlates

This example describes a nucleic acid hybridization assay carried out ona plate manufactured as described for Plate G in Example 6.2. Theexposed surface of the working electrode in each well was coated bydispensing on the working electrode 1,500 nL of a solution containingavidin at a concentration of 1 mg/mL and allowing the solution to dry onthe surface (the avidin was confined to the working electrode surface bythe fluid barrier provided by the conductive tape). After drying theavidin solution on the working electrode, the excess unbound antibodywas removed (and uncoated surfaces blocked) by filling the wells with asolution containing 5% (w/v) bovine serum albumin (BSA) in phosphatebuffered saline (PBS). The plates were incubated with the blockingsolution overnight at 4° C. and then washed with PBS.

Avidin-coated plates provide a convenient generic platform for theimmobilization of biotin-labeled reagents. In this example, abiotin-labeled 28 nucleotide DNA probe sequence was immobilized on theplates by introducing to each well 0.05 mL of a 100 nM solution of thebiotin-labeled probe, incubating the plate for 1 hour while shaking on aplate shaker and washing excess probe away with PBS. The immobilizedprobe was used to assay for samples containing a complementary DNAtarget sequence that was labeled at the 5′ position with a derivative ofruthenium-tris-bipyridine (TAG Phosphoramidite, IGEN International).Varying amounts of the labeled target DNA sequence in a volume of 0.05mL were introduced into the wells and allowed to hybridize at roomtemperature over a period of 1 hour while shaking the plate on a plateshaker. After washing the wells with PBS, the wells were filled with 0.1mL of ORIGEN Assay Buffer (IGEN International) and analyzed as describedin Example 6.3. FIG. 31 shows that the ECL signal (corrected for thebackground signal observed in the absence of the labeled targetsequence) was linearly dependent on the concentration of the targetsequence over a concentration range exceeding four orders of magnitude.

6.8 Use of Multi-Well Assay Plates and Luminescence Imaging Apparatus inChemiluminescence-Based Assays

This example describes a chemiluminescence-based binding assay carriedout on a plate manufactured as described for Plate B in Example 6.2. Inthis example, the carbon ink working electrode of the plate is not usedas an electrode but is only used as a high surface area solid phasesupport for binding reagents. The exposed surface of the workingelectrode in each well was coated by dispensing on the working electrode2,500 nL of a solution containing avidin at a concentration of 1 mg/mLand allowing the solution to dry on the surface (the avidin was confinedto the working electrode surface by the fluid barrier provided by theconductive tape). After drying the avidin solution on the workingelectrode, the excess unbound antibody was removed (and uncoatedsurfaces blocked) by filling the wells with a solution containing 5%(w/v) bovine serum albumin (BSA) in phosphate buffered saline (PBS). Theplates were incubated with the blocking solution overnight at 4° C. andthen washed with PBS.

The avidin-coated plates were used as a solid phase for assaying for abiotin labeled antibody. Samples containing varying amounts of abiotin-labeled mouse monoclonal IgG in 0.05 mL of 0.1% BSA/PBS wereadded to the wells. The plates were mixed on a plate shaker for 1 hourand then washed with PBS. The wells were then treated with 0.05 mL of a1:10,000 dilution of a alkaline phosphatase labeled goat anti-mouseantibody (Sigma) diluted in 0.1% BSA in PBS. The plates were mixed on aplate shaker for 1 hour and then washed with PBS followed by a Trisbased buffer. A solution containing a chemiluminescent alkalinephosphate substrate and a chemiluminescence enhancer (50 uL of EmeraldII substrate and enhancer, Perkin Elmer) was added to the wells. Theplate was allowed to incubate for 5-10 min to allow the chemiluminescentreaction to stabilize and was then imaged using the imaging plate readerdescribed in Example 6.3 except that an electrical potential was notapplied to the plates. FIG. 32 shows the chemiluminescence as a functionof the concentration of biotin-labeled monoclonal antibody. Thechemiluminescence was considerably more intense than that measured in ananalogous experiment using avidin adsorbed on standard polystyreneplates; presumably, the assay on the carbon surface benefited from thehigh surface area and excellent adsorptive properties of theplasma-treated carbon surface.

6.9 ECL Measurements in 1536-Well Plates

The basic multi-well plate structure shown in FIG. 8A was adapted to a1536-well plate format. The sectioned working electrode layer was madeby screen printing, on a Mylar substrate, 6 square pads composed ofcarbon ink over silver ink. The conductive pads were connected toscreen-printed electrical contacts on the back of the plate (also carbonink over silver ink) through laser-cut through-holes in the Mylarsubstrate that filled with conductive material during thescreen-printing steps. The counter electrode layer was made bypatterning 2-mil thick aluminum foil using standard photolithographictechniques to produce a 48×32 array of square holes in the foil: i) thefoil was coated with a layer of photoresist, ii) the photoresist waspatterned by illumination through a patterned mask, iii) the photoresistwas washed to reveal a pattern of exposed aluminum, iv) the aluminum waschemically etched to produce the array of holes and v) the remainder ofthe photoresist was removed. By analogy to FIG. 8A, the aluminum filmwas oversized relative to the plate so as to allow it to be foldedaround the working electrode layer. This oversized section of thealuminum foil had photolithographically defined holes so as to allowcontact through the aluminum film to the working electrode contacts. Theetched aluminum foil was then laminated on one side to a dielectric filmhaving the same pattern of holes.

The plate was assembled as follows. Double sided adhesive tape having alaser-cut 48×32 array of square shaped holes (in the 1536-well pattern)was aligned and mated to the bottom of a 1536-well plate top with squarewells (Greiner America). The remaining exposed side of the adhesive tapewas then aligned and mated to the non-laminated side of the patternedaluminum foil. A second layer of double sided adhesive tape with alaser-cut 48×32 array of square holes was then aligned and mated to thelaminated side of the aluminum foil. Finally, the remaining exposed sideof the second layer of double sided adhesive was aligned and mated to aMylar substrate so as to form a 1536-well plate with wells having wallsdefined by holes through the plate top, the laminated aluminum foil andthe two layers of double sided adhesive, and having well bottoms definedby the working electrode pads. To complete the plate structure, a thirdlayer of double sided adhesive tape (this layer having holes patternedin the same arrangement as the oversized section of the aluminum foillayer) was aligned and mated to the back of the screen-printed Mylarsubstrate. The oversized section of the laminated aluminum foil layerwas folded back around the substrate and mated to this third layer ofdouble sided adhesive tape so as to allow electrical contact to thealuminum foil as well as electrical contact (through the holes in thelaminated aluminum foil and the third layer of double sided adhesivetape) to the patterned electrical contacts on the back of the substrate.

ECL was induced in and measured from the 1536-well plates was read onthe instrument designed to make electrical contact to individual squaresectors (as described in Example 6.3). Solutions containing varyingconcentrations of ruthenium(II)-tris-bipyridine dichloride in aTPA-containing buffer (ORIGEN Assay Buffer, IGEN International) weredispensed into wells of a plate (0.005 mL/well). ECL was induced byramping the voltage applied to the working electrode from 2 to 4 voltsover a period of 3 seconds. FIG. 33 shows the integratedelectrochemiluminescence associated with each well as a function of theconcentration of ruthenium(II)-tris-bipyridine dichloride in the well.Each point represents the average of values obtained from 32 wells ofthe plate. The expected linear dependance of ECL intensity with theconcentration of ruthenium(II)-tris-bipyridine dichloride was observed(slope of log-log plot as determined by linear regression=1.06).

Section 6.10: Effect of Type of TiO₂ on Photochemically InducedLuminescence

The following forms of TiO₂ were tested to determine the effect of typeof TiO₂ on photochemically induced luminescence. All grades are rutileexcept where noted:

Grade Source Inorganic treatment Organic treatment R101 DuPont 1.7%alumina 0.2% polyol R102 DuPont 3.2% alumina 0.25% polyol R104 DuPont1.7% alumina 0.3% silicone R105 DuPont 2.5% alumina, Yes, butundisclosed 3.0% silica R960 DuPont 3.5% alumina, none 6.5% silica RCL 6Millienium Silica Yes, but undisclosed RCL 188 Millienium Phosphate Yes,but undisclosed Anatase Millienium Unknown Unknown

Experiment 1

Procedure:

-   -   1. 0.6 grams of each type of TiO₂ in powder form were weighed        out.    -   2. 3.4 grams of epoxy were weighed out and mixed by hand with        the TiO₂ to give a final concentration of 15 weight percent        TiO₂.    -   3. All the samples were then spotted onto an aluminum surface.    -   4. The surface was exposed to either UV or fluorescent light for        several seconds and then placed in a reader with a CCD camera.    -   5. The luminescent intensity was read after 15 seconds.

Results: 15 wt. % TiO₂ in epoxy - 15 seconds after insertion intoinstrument Background corrected light intensity Grade Source UV lightFluorescent R101 DuPont 19 22 R102 DuPont 28 36 R104 DuPont 13 14 R105DuPont 15 17 R960 DuPont 13 12 RCL6 Millienium 38 44 RCL 188 Millienium86 96 Anatase Millienium — 25

From this experiment, the alumina surface treatment (DuPont grades)appears to reduce light emission 4-5 times compared to phosphatetreatment (RCL 188) and by 2-3 times compared to silica (RCL 6).

The two best grades R104 and R960 and the RCL 188 were compounded intopolystyrene to see if the effect was the same in the desired polymer(Experiment 2).

Experiment 2

The above experiment was repeated for three forms of the TiO₂ testedabove compounded into polystyrene at ˜5 and 15 weight percent using atungsten light source. Samples were measured in triplicate. The lightintensity was read at 15 seconds after insertion into the instrument.

Results in polystyrene - 15 seconds after insertion into instrumentBackground corrected light intensity Concentration Standard Grade (wt.%) Mean Deviation R104 16 14 2 R104 5 30 7 R960 19 16 1 R960 6 6 1RCL188 16 84 19 RCL188 5 10 1 Polystyrene 0% TiO₂ 0 0Again, the alumina coated TiO₂ emitted less light than the silica coatedmaterial. For R104, the emitted light decreased with increased TiO₂concentration. The experiments were repeated with the same results. Theconcentration of TiO₂ for the R104 samples was verified by anindependent measurement.

7. Incorporation of References

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingfigures. Such modifications are intended to fall within the scope of theclaims. Various publications are cited herein, the disclosures of whichare incorporated by reference in their entireties.

The invention claimed is:
 1. An apparatus for measuring luminescencefrom a multi-well assay plate having a plurality of wells, saidapparatus comprising: (a) an imaging system comprising a detector, theimaging system configured to image one or more sectors of said pluralityof wells in said multi-well assay plate and measure luminescence emittedtherefrom, wherein the detector is selected from the group consisting ofa camera, a photodetector, a photodiode, an avalanche photodiode, acharged-coupled device (CCD) chip, a CCD camera, a photomultiplier tube,a complementary metal-oxide-semiconductor (CMOS) detector, a film, aphosphorescent material, and an intensifier; and (b) a plurality ofelectrical connectors adapted to connect a source of electrical energyto said one or more sectors of said plurality of wells, wherein saidelectrical connectors are positioned on an electrical connector surfaceand configured to substantially vertically align said plurality ofelectrical connectors with a bottom surface of said multi-well assayplate and contact said bottom surface of said multi-well plate.
 2. Theapparatus of claim 1, wherein said electrical connector surface isconfigured to substantially vertically align said plurality ofelectrical connectors, said one or more sectors of said plurality ofwells and said imaging system.
 3. The apparatus of claim 1, comprisingtwo or more electrical connectors.
 4. The apparatus of claim 1, furthercomprising a multi-well plate holder and said electrical connectors arepositioned on a top surface of said plate holder.
 5. The apparatus ofclaim 1, wherein said plurality of electrical connectors comprise one ormore working electrical connectors and two or more counter electricalconnectors.
 6. The apparatus of claim 1, wherein bottom surfacecomprises one or more contact surfaces and said plurality of electricalconnectors are configured to contact said plate bottom at said one ormore contact surfaces.
 7. The apparatus of claim 1, wherein said imagingsystem further comprises a mounting system for positioning the detectorrelative to said one or more sectors of said plate.
 8. The apparatus ofclaim 1, further comprising one or more additional subsystems including:(i) a plate translation subsystem; (ii) a plate shaking subsystem; (iii)a plate storage subsystem; (iv) a reagent handling subsystem; (v) areagent delivery subsystem; and (vi) combinations thereof.
 9. Theapparatus of claim 1, wherein said detector is a CCD camera or chip. 10.The apparatus of claim 1, wherein said detector is a photodiode oravalanche photodiode.
 11. The apparatus of claim 1, wherein saiddetector comprises an array of photodiodes.
 12. A multi-well assay platehaving a plurality of wells, said plate comprising: a plurality ofelectrical contacts supported on a bottom surface of said multiwellplate, each electrical contact is adapted to connect a source ofelectrical energy to, and substantially vertically aligned with, one ormore sectors of said plurality of wells.
 13. The plate of claim 12,wherein the plate is configured for imaging by an imaging systemcomprising a detector, the imaging system configured to image said oneor more sectors of said plurality of wells in said multi-well assayplate and measure luminescence emitted therefrom, wherein the detectoris selected from the group consisting of a camera, a photodiode, anavalanche photodiode, a charged-coupled device (CCD) chip, a CCD camera,a photomultiplier tube, a complementary metal-oxide-semiconductor (CMOS)detector, a film, a phosphorescent material, and an intensifier.
 14. Theplate of claim 13, wherein said bottom surface of said multiwell plateis configured to substantially vertically align said plurality ofelectrical contacts, said one or more sectors of said plurality of wellsand said imaging system.
 15. The plate of claim 12, comprising two ormore electrical contacts.
 16. The plate of claim 12, further comprisinga multi-well plate holder and said electrical contacts are positioned ona top surface of said plate holder.
 17. The plate of claim 12, whereinsaid plurality of electrical contacts comprise one or more workingelectrical contacts and two or more counter electrical contacts.
 18. Theplate of claim 12, comprising seven electrical contacts.
 19. The plateof claim 12, wherein said bottom surface comprises one or more contactsurfaces and said plurality of electrical contacts are configured tocontact said plate bottom at said one or more contact surfaces.
 20. Theplate of claim 13, wherein said imaging system further comprises amounting system for positioning the detector relative to said one ormore sectors of said plate.
 21. The plate of claim 13, furthercomprising one or more additional subsystems including: (i) a platetranslation subsystem; (ii) a plate shaking subsystem; (iii) a platestorage subsystem; (iv) a reagent handling subsystem, (v) a reagentdelivery subsystem; and (vi) combinations thereof.
 22. The plate ofclaim 13, wherein said detector is a CCD camera or chip.
 23. The plateof claim 13, wherein said detector is a photodiode or avalanchephotodiode.
 24. The plate of claim 13, wherein said detector comprisesan array of photodiodes.
 25. The plate of claim 12, wherein saidelectrical contacts are spring-loaded.
 26. The plate of claim 12,wherein each of said plurality of wells comprises an inner bottomsurface.
 27. The plate of claim 26, further comprising a plurality ofelectrodes, wherein each inner bottom surface of each of said pluralityof wells comprises an electrode of the plurality of electrodes.
 28. Theplate of claim 27, wherein each of said plurality of electrodes areconnected to a portion of the bottom surface of said multiwell plate.29. The plate of claim 12, wherein the plurality of electrical contactsare configured to receive electrical energy from the electricalconnectors of claim
 1. 30. The apparatus of claim 1, wherein saidelectrical connectors are positioned on, and extend a distance from, theelectrical connector surface.